Ethnomedicinal appraisal of plants used for the treatment of gastrointestinal complaints by tribal communities living in Diamir district, Western Himalayas, Pakistan.

Majority of the mountain dwelling communities living in the Himalayas rely on traditional herbal medicines for primary healthcare needs. Present study was conducted in fairy meadows and allied valleys in District Diamir, Gilgit Baltistan autonomous territory in northern Pakistan. Documentation of traditional medicinal knowledge (TMK) of local communities for the treatment of gastrointestinal disorders was carried out as a component of a wider medico-botanical expedition conducted in the entire base camp of the great Nanga Parbat peak during 2016-19. Various ethnobotanical parameters i.e. use value (UV), informant consensus factor (ICF), Fidelity level (FL), direct matrix ranking test (DMRT) and preference ranking (PR) were applied to evaluate the data collected during field surveys. The plants were also subjected to a comparative review for novelty assessment. A total of 61 medicinal plant species belonging to 55 genera and 35 families are reported here for the treatment of GIDs. Compositae was the leading family with 8 (13%) species. Fourteen gastrointestinal disorders were cured with 32% taxon were reported for stomachic followed by diarrhea (15%) and constipation (14%). Highest use reports (5) and use citations (207) were reported for Mentha longifolia L. while highest UV (1.79) was obtained for Artemisia maritima L. Hylotelephium telephioides (Ledeb.), A. maritima, M. longifolia, M. piperita L., Allium cepa L., and A. annua L. exhibited 100% FL. Highest ICF was calculated against dysentery and flatulence. DMRT ranked Prunus persica L. first for its multipurpose uses. Taking constipation as a reference gastrointestinal disease, PR for ten plant species was calculated where H. telephioides was ranked first followed by A. maritima. Present study concluded that 19 out of 61 plant species were documented for the first time with novel medicinal uses to cure GIDs. These plant species could act as potential reservoirs of novel lead compounds for the treatments of gastrointestinal disorders.

Introduction

More than 70% of rural population in developing countries relies on traditional medicinal plants for their primary health needs [1]. Digestive track ailments, collectively known as gastrointestinal disorders (GIDs) are cited more frequently in mountain dwelling communities where poor sanitation practices are more common [2]. GIDs range from short-term indigestion or flatulence to long-term structural anomalies and chronic illnesses having a substantial influence on morbidity and mortality. According to world health organization (WHO) estimates, GIDs, caused nearly 1 million adult deaths worldwide during 2019 where diarrhea alone was responsible for 370,000 death in children under the age of 5 years [3]. Apart from mortality and morbidity, GIDs have some intrinsic relationship with overall human wellbeing. Recent data has suggested that some GIDs act as major contributing factor in onset of neurological disorders such as Parkinson’s Disease and Alzheimer’s Disease [4]. Botanicals formulations and plant extracts are recently being investigated as modulators for “brain-gut-microbiota axis” a recently adopted phrase for emerging interface of human ailments [5, 6]. Use of medicinal plants for GIDs has extensively been reported from ethnobotanical expedition across the world since few decades [7-12]. Field surveys have reported that instant relief from gut ailments strengthens the trust of locals on plant derived home remedies as alternative to costly and slowly acting allopathic drugs [13, 14]. Moreover, many of the GIDs are considered taboos in conservative Alpine Asian culture. Bad breath, Piles, irritable bowel syndrome, gas and many other GIDs are never shared comfortably with a physician, who, in most of the cases would be a non-native person [15, 16]. Either way, the trust of indigenous people on traditional medicine used for GIDs is exceptionally high. On the other hand, plant-based therapies are the only choice for rural communities in various remote areas of third world countries including Pakistan. Tribal communities such as those living in vales and river terraces of Karakorum and Himalayas conserve a very unique assemblage of plant use practices [17]. Gilgi-Baltistan along with adjoining Chinese areas represent a greater biodiversity hotspot hosting more than 300 medicinal and aromatic plants [18, 19]. Recently, a couple of targeted medico-botanical expeditions have revealed various medicinally important species of genus Berberis are documented with extensive use practices [20, 21]. Owing to ethnic diversity, and historical linkages with various civilizations, entire region of Gilgit-Baltistan is very rich in traditional medicinal practices [22].For centuries, the area has acted as a pedestrian crossroad between mainland China, Central Asia, and Indian subcontinent. This has led to a blending of three greater civilizations [23-25]. That is why ancient ayurvedic wisdom shares many commonalities with Traditional Chinese medicine (TCM) [26]. There are certain geographic limitations that impose a sustainable food-cum-medicine use strategy of indigenous flora as an imperative of survival in almost all areas of upper Indus basin (UIB) including District Diamer [27].

The study area comprises of the “Fairy Meadows” and allied tribal setting at the base camp of Nanga Parbat, the second highest peak in Pakistan. Fairy Meadows, locally known as “Joot”, was named by German mountaineers who got mesmerized on their maiden journey to the areas. It was declared a national park in 1995 as an acknowledgement of diverse flora, fauna, and geographic features. There is a large number of endemic species though not documented very well for their taxonomic status and medicinal uses [28]. The road leading to study areas is declared as world’s second most dangerous track; that speaks of its remoteness and staying largely unexplored yet. More than 15 km of jeep ride is required for mere entry in to the ‘Fairy Meadows” while rest of the study sites lie on small river-let basins and ascending terraces towards south-western and northern faces of Nanga Parbat. Health infrastructure is next to nothing in the area that implies almost complete reliance of the inhabitants of locally available healing ways largely comprised of plant derived home recipes or formulations prescribed by locally practicing herbal healers.

Materials and methods

Statement of ethics

The field study was conducted in strict compliance to ethical standards for ethno botanical field studies. Formal ethical approval was acquired from institutional ethics committee vide.

Filed surveys

After preliminary survey, a series of target expeditions were conducted in different villages and seasonal settlement across alpine landscape during summers. A detailed inventory of medicinal plants was developed during April 2018 to July 2019. Structured and semi-structured interviews were conducted for the ethnobotanical data collection. Informant consent and consent for the publication was taken from the elders of the community before the interviews.

Study area

District Diamir shares boundaries with Khyber Pakhtunkhwa (KPK) province in the south, Azad Kashmir in the west, Ghizer District in the east and Astore district in the north. The study area has small villages, having human settlements namely Buner Das, Halaly, Thamros, Gashot, Pakora and some small patches with human settlements. Extreme summer temperature rises up to 38°C and winter temperature fall below 0°C in the valleys [29]. The Nanga Parbat (ninth-highest peak on the earth measuring 8126 m ASL) is the western anchor of Himalayan ranges known as the last eight thousander in the mighty mountain range. The study sites are situated between 35°23’14.3"N latitude and 74°34’44.7"E longitude, at an elevation range between 934–7937 m above the sea level (). District Diamir covers an area of 10936 Km2, divided into two tehsils which are: tehsil Chilas and tehsil Darel/Tangir. Gilgit Baltistan has an important strategic location as it hosts the confluence of three great world mountainous ranges, the Karakorum, the Hindu Kush and the Himalayas, providing the habitat for at least 10% of global flora [30].

Participants of the study area

The study area has a rich diversity of culture and ethnic groups. Different languages like Shina, Gojri, Kashmiri and Dulasgariya are spoken in the area. Shina is the dominant language as 100% of the studied population can speak and understand it. Various ethnic groups like Sheen, Youshkon, Gujar, Dulasgar and Kashmiri reside in the study area. A total of 166 individuals including 115 men and 51women were interviewed about the use of selected medicinal plants for the treatment of GIDs (Table 1).

Table 1

Demographics of the study area.

Demographic feature	Criteria	Number of informants	Percentage
Gender of informants	Male	115	69.3
	Female	51	30.7
Distribution by Village	Buner Das	27	16.3
	Thamros	40	24.1
	Gashot	36	21.7
	Pakora	30	18.1
	Keli Jail	15	9
	Halaly	18	10.8
Age of Informants	Between 20 to 30	16	9.6
	Between 31 to 40	23	13.9
	Between 41 to 50	39	23.5
	Between 51 to 60	45	27.1
	Above 60	43	25.9
Marital status	Married	123	74.1
	Unmarried	36	21.7
	Widow	7	4.2
Education level	Illiterate	97	58.4
	Elementary School	25	15.1
	Secondary School	22	13.3
	College	15	9
	University	7	4.2
Social Livelihood	Farmer	112	67.5
	Retired	5	3
	Shepherd	31	18.7
	Others	18	10.8
Residence	Village	155	93.4
	Seasonal migrants	11	6.6
Ethnic group	Youshkon	79	47.6
	Sheen	34	20.5
	Gujar	38	22.9
	Kashmiri	15	9
Experience	Herbalists	7	4.2
	Local people	159	95.8
Duration of residence in the area	Less than 15 years	9	5.4
surveyed area	More than 15 years	157	94.6
Religion	Islam	166	100

Documentation of ethnomedicinal data

Ethnomedicinal data was documented using questioners (structured and semi-structured) interviews, participant observations, guided field walks, and focus group discussions [31-33]. Information like local name of the plant, plant parts used, recipe, disease treated, and other information were collected from locals. In addition to this, standard data collection methods [34] have been followed for the documentation of indigenous knowledge of community on health, use, conservation and threats of medicinal plants. Prior rural consent and consent for the publication was obtained from the elders (Lumberdar) orally and the ethical standards of the Society for Economic Botany and International Society of Ethnobiology [35] were followed. All the interviews were conducted in local Shina language.

Plant collection and identification

Following the interviews and discussions, voucher specimens were collected. These specimens were pressed and mounted on herbarium sheets. Botanical identification of the specimens was done by Prof. Dr. Rahmatullah Qureshi, at Pir Mehr Ali Shah University of Arid Agriculture Rawalpindi (PMAS UAAR) Pakistan, and authenticated with the help of flora of Pakistan (http://www.efloras.org/flora_page.aspx?flora_id=5), The Plant List (http://www.theplantlist.org/), International Plant Name Index (https://www.ipni.org/) and World Flora Online (http://www.worldfloraonline.org/) with already identified specimens. Air-dried specimens were systematically tagged, labeled and voucher number for each specimen was allotted (Table 2). The identified specimens were deposited in the herbarium of Department of Botany, PMAS UAAR for future reference.

Table 2

Plant species with accession ids, GIDs treated, citations, frequency of citation (FC), relative frequency of citation (RFC) and use value (UV).

S.No.	Local Name	Taxonomic Name	Uses	UR	Use citations	FC	RFC	UV
1	Zoon	Artemisia maritima L./RW735	Intestinal worms (65), diarrhea (43), vomiting (71) & stomachic (20)	4	200	112	0.67	1.79
2	Philil	Mentha longifolia (L.) L./RW745	diarrhea (89), intestinal worms (41), digestive disorders (23), vomiting (44) & stomachic (10)	5	207	140	0.84	1.48
3	Nerlay Zoon	Tanacetum faconeri Hook.f./RW739	diarrhea (89), vomiting (53), stomachic (47)	3	189	132	0.80	1.43
4	Philil	Mentha piperita L./RW736	Diarrhea (79), indigestion (21) & ulcer (9)	3	109	99	0.60	1.10
5	Kasho/Paloan	Allium cepa L./RW744	Diarrhea (51), vomiting (34)	2	85	78	0.47	1.09
6	Choro	Pimpinella diversifolia DC./RW746	Stomachic (42), Intestinal worms (38)	2	80	86	0.52	0.93
7	Teetar	Hylotelephium telephioides (Ledeb.) H. Ohba/RW741	Constipation (132)	1	132	143	0.86	0.92
8	Chontal	Rheum webbianum Wall./RW747	Stomachic (31), constipation (18) & intestinal worms (21)	3	70	76	0.46	0.92
9	Nerlay Churki	Oxyria digyna (L.) Hill/RW740	diarrhea (67) & digestive (36)	2	103	112	0.67	0.92
10	Lilio	Viola serpens WalL./RW748	diarrhea (23), constipation (11) & stomachic (23)	3	57	62	0.37	0.92
11	Shey Lamay	Persicaria amplexicaulis (D.Don) Ronse Decr./RW749	Stomach disorders (57)	1	57	62	0.37	0.92
12	Cheti Char	Cichorium intybus L./RW750	Ulcer (22), stomachic (43)	2	65	79	0.48	0.82
13	Aaro	Prunus persica L./RW751	Intestinal worms (21), stomachic (26) & gastritis (9)	3	56	69	0.42	0.81
14	Angrezi phang	Ficus carica L./RW752	Constipation (24) & stomachic (31)	2	55	68	0.41	0.81
15	Joi	Prunus armeniaca L./RW753	Diarrhea (32) & constipation (21)	2	53	67	0.40	0.79
16	Love	Cucumis sativus L./RW754	Digestive (29) & purgative (30)	2	59	77	0.46	0.77
17	Tumurum	Thymus serphyllum L./RW755	Stomachic (75)	1	75	98	0.59	0.77
18	Chenga	Persicaria vivipara (L.) Ronse Decr./RW756	diarrhea (3) & constipation (43)	2	46	61	0.37	0.75
19	Goom	Triticum aestivum L./RW757	Piles (56)	1	56	76	0.46	0.74
20	Bushi punar	Saussurea gossypiphora D.Don/RW742	constipation (54)	1	54	75	0.45	0.72
21	Susar	Rhododendron anthopogon D. Don/RW758	Stomachic (74)	1	74	104	0.63	0.71
22	Patrees	Aconitum heterophyllum Wall. ex Royle/RW759	Stomachic (27) & intestinal worms (16)	2	43	61	0.37	0.70
23	Jomi	Urtica dioica L./RW760	Vomiting (31)	1	31	44	0.27	0.70
24	Koret	Bergenia stracheyi (Hook.f. & Thomson) Engl./RW761	Stomachic (47) & diarrhea (24)	2	71	102	0.61	0.70
25	Simbul Char	Adiantum raddianum C. Presl/RW762	diarrhea (41)	1	41	59	0.36	0.69
26	One	Cucurbita maxima Duchesne/RW763	Stomachic (27) & constipation (16)	2	43	62	0.37	0.69
27	Shatoo	Ribes alpestre Wall.ex Decne./RW764	Ulcer (22)	1	22	32	0.19	0.69
28	Margosh Chontal	Rheum australe D. Don/RW743	Intestinal worms (64) & constipation (32)	2	96	142	0.86	0.68
29	Peban Maroch	Morus alba L./RW765	Stomachic (29) & constipation (21)	2	50	74	0.45	0.68
30	Mulo	Raphanus sativus L./RW766	Stomachic (33)	1	33	50	0.30	0.66
31	Gizari	Daucus carota L./RW767	Digestive (43)	1	43	67	0.40	0.64
32	Nooni Char	Oxalis corniculata L./RW768	Constipation (23) & ulcer (11)	2	41	65	0.39	0.63
33	Chorko	Berberis lycium Royle/RW769	Stomach problem (41)	1	41	65	0.39	0.63
34	Konay	Echinops echinatus Roxb./RW770	Abdominal pain (22)	1	22	35	0.21	0.63
35	Churki	Rumex hastatus D. Don/RW771	Stomachic (29) & flatulence (22)	2	51	82	0.49	0.62
36	Hailel	Solanum nigrum L./RW772	Dysentery (9), stomachic (23) & ulcer (8)	3	40	65	0.39	0.62
37	Khakao	Pistacia khinjuk Stocks/RW773	indigestion (41)	1	41	67	0.40	0.61
38	Khaneray Char	Salvia sp./RW737	Stomachic (71)	1	71	118	0.71	0.60
39	Hamay	Dysphania botrys (L.) Mosyakin & Clemants/RW774	Abdominal pain (6) & diarrhea (17)	2	23	41	0.25	0.56
40	Bhendi	Abelmoschus esculentus (L.) Moench/RW775	Digestive (27)	1	27	49	0.30	0.55
41	Hayao	Bunium persicum (Boiss) B. Fedtsch./RW776	stomachic (41)	1	41	76	0.46	0.54
42	Makai	Zea mays L./RW777	Stomachic (32) & gastritis (27)	2	59	111	0.67	0.53
43	Tandur	Datura stramonium L./RW778	Stomachic (32)	1	32	61	0.37	0.52
44	Shangali	Cuscuta reflexa Roxb.	Stomach disorders (23)	1	23	44	0.27	0.52
45	Shaftal	Trifolium repens L./RW737	Stomachic (22)	1	22	43	0.26	0.51
46	Bangra	Swertia petiolata D. Don/RW738	Stomachic (41)	1	41	81	0.49	0.51
47	Khapoy Patay	Plantago himalaica Pilg./RW779	Stomachic (19) & diarrhea (9)	2	28	59	0.36	0.47
48	Chilli	Juniperus excelsa M.Bieb./RW780	diarrhea (43)	1	43	92	0.55	0.47
49	Danoi	Punica granatum L./RW781	Intestinal worms (32)	1	32	72	0.43	0.44
50	Zooti Ponar	Aster himalaicus C. B. Clarke/RW782	Stomachic (18)	1	18	44	0.27	0.41
51	Kasheel/Zach	Vitis vinifera L./RW783	Constipation (37) & intestinal worms (3)	2	40	98	0.59	0.41
52	Gulab	Rosa indica L./RW784	Stomachic (19) & constipation (12)	2	31	78	0.47	0.40
53	Kuna	Chenopodium album L./RW785	Constipation (12) & abdominal pain (5)	2	17	44	0.27	0.39
54	Aseel Khukunay	Cicer microphyllum Benth./RW786	Indigestion (5) & vomiting (9)	2	14	39	0.23	0.36
55	Loi Margan	Capparis spinosa L./RW787	Piles (7) & digestive (5)	2	12	35	0.21	0.34
56	Kino Maroch	Morus nigra L./RW788	Stomachic (21)	1	21	63	0.38	0.33
57	Hazar Daru	Limonium cabulicum (Boiss.) Kuntze/RW789	Stomachic (21)	1	21	75	0.45	0.28
58	Dadi Pushi Char	Xanthium strumarium L./RW790	Ulcer (11)	1	11	43	0.26	0.26
59	Pharphara	Verbascum thapsus L./RW791	diarrhea (11)	1	11	54	0.33	0.20
60	Ishpit	Medicago sativa L./RW792	Stomachic (12)	1	12	65	0.39	0.18
61	Khakos	Artemisia annua L./RW793	diarrhea (3) & vomiting (4)	2	7	58	0.35	0.12

Data analysis

The ethnomedicinal data obtained during the field surveys was shifted to the Microsoft excel spread sheet and organized in a tabulated form for presentation. Various quantitative ethnobotanical indices like Use Value (UV), Frequency of Citation (FC), Relative Frequency of Citation (RFC), Direct Matrix Ranking (DMR), Fidelity Level percentage (FL), and Informant Consensus Factor (ICF) were applied for the graphical representation of the numerical data extracted.

Relative frequency of citation

The RFC was determined following [36] which expresses the local importance of the plant species. RFC was calculated as a measure of citation frequency that explains the intensity of uses reported for a given species by all the informants It was calculated by dividing the frequency of citation (FC) by the total number of informants involved in the survey (N) as shown below: Where FC is the frequency of citation which denotes the number of informants interviewed for a species who site its use while N is the total number of informants involved in the survey.

Use value

Use value (UV) expresses the relative importance of the medicinal plant species based on number of recorded uses for each species, known locally. UV is considered a baseline quantitative index in ethnobotanical studies. It was calculated by the methodology given by [37], using following formula: Where UV stands for the use value; U refers to the total number of uses for each species; and ‘‘n” is the total number of informants who reported that species.

Fidelity level (FL)

Fidelity level explains the specific use of each plant species and its preference over other species by expressing the specificity of disease treated by a reported plant species. It is applied to distinguish the crucial role of reported species. It is calculated by a formula [38] which is as follows: Where “FL” is the percent fidelity level; “Ip” is the number of informants who mentioned the plant species for the treatment of a particular disease; and “Iu” is the number of informants who mentioned the same plant for any other use.

Informant consensus factor (ICF)

Informant consensus factor was calculated by following the formula given by [39]. It expresses the consensus of the informants about the use of plant species for the treatment of various diseases. Where “Nur” is the number of use reports for a disease treated by a plant species while “Nt” is the number of plant taxa used to treat that disease. ICF varies from 0–1. It is used to estimate the significance of each medicinal use category centered on the uniformity of the interviewer’s response.

Direct matrix ranking (DMRT)

Direct matrix ranking test (DMRT) was calculated following the method of [32]. It was conducted to compare the species mentioned for multiple uses by the informants. Based on the relative benefits obtained from each plant, ten multipurpose plant species were selected, and seven use diversities of these plants were listed. Five key informants were chosen to assign use values for each attribute (4 = best, 3 = very good, 2 = good, 1 = less, 0 = none). The use categories included medicinal use, fuel, construction, edible, agriculture tools, thatching, fodder, and forage. Based on data obtained from the informants, the average use diversity value for each species was determined and the values for each species were finally summed and ranked.

Preference ranking (PR)

Ten informants were randomly selected to assess the degree of effectiveness of medicinal plant species when used to treat constipation, a gastrointestinal disease category following the methodology given by [40]. The medicinal plants believed to be most effective to treat constipation were given the highest value (5), while the least effective received the lowest value (1). The value of each species was summed and the rank for each species was determined based on the total score. This helped to indicate the most effective medicinal plants used by the informants to treat a serious gastrointestinal disease (constipation).

Novelty assessment. Novelty assessment was the prime objective of present study because it was very likely that the study area owing to its remoteness and peculiar tribal composition would be harboring unique plant use pracitces. From the selected plant species, a few novel plant species were identified with novel use reports from the study area. These species were subjected with a robust comparative review using literature databases viz; google scholar, PubMed and Scifinder to explore any previously reported pharmacological and ethnomedicinal uses of these plants and to compare with the use reports mentioned by locals in present study.

Results

Sociodemographic characteristics of the respondents

One hundred and sixty-six (166) informants were interviewed during the ethnomedicinal inventory. Of which, 151 comprising up of 69% were males followed by 51 contributing 31% females. Of these, 40(24%) informants were selected from Thamros village, 36(22%) from Gashot, 30(18%) from Pakora, 27(16%) from Buner Das, 18(11%) from Halaly, and 15(9%) from Keli Jail. Age wise, 10% of the informants had 20 to 30 years of age, 14% had 31–40 years, 24% had 41–50 years, 27 had 51–60 years and 26% of the informants had above 60 years of age. It was observed that age of the informants greatly affects the ethnomedicinal information as the older people have more experience and knowledge in this regard. The marital status of the informants was also noted. It was observed that 123(74%) of the informants were married and 36(22%) were unmarried while 7(4%) were widowed.

Majority of the informants were illiterate 97(58%) followed by elementary school 25(15%), secondary school 22(13%), college 15(9%) and university students 7(4%). Due to remoteness of the study area, majority of the people rely on agriculture and livestock practices for their livelihood. Out of 166, 112(68%) of the informants were farmers which is followed by shepherd 31(19%), retired persons 5(3%) and others 18(11%). Most of the people 155(93%) were residing in villages and only 11(7%) were seasonal migrants who live both in villages and cities in different seasons of the year. Four different ethnic groups were residing in the study area among which 79(48%) were youshkon, 34(20%) sheen, 38(23%) gujar and 15(9%) were Kashmiri. From the study area, 7(4%) of the informants have experience in herbal medications and they were practicing in cities as herbalists and 159(96%) have no proper experience in herbalism and they self-medicate of their illness with medicinal plants. A very few 9(5%) informants were residing in the study area from less than 15 year while 157(95%) were living there from more than 15 years. Religiously, 100% of the informants were Muslim (Tab.1)

Diversity of medicinal plants for treatment of gastrointestinal disorders

A total of 61 medicinal plant species belonging to 55 genera and 35 families have been documented from the study area for the treatment of GIDs. Compositae was the most dominant family with 8(13%) species followed by Polygonaceae 6(10%), Lamiaceae, Leguminosae, Moraceae and Rosaceae 3(5%) each, Apiaceae, Cucurbitaceae, Poaceae and Solanaceae 2(3%) each and all other species were contributing 1(2%) ( Fourteen different gastrointestinal disorders have been treated by the collected medicinal plants of which stomach disorder was the leading as 33(54.1%) plant species were used for its treatment followed by diarrhea 16(26.2%), constipation 14(23%), intestinal worms 9(14.8%), vomiting 7(11.5%), digestive and ulcer 6(9.8%) each, indigestion and abdominal pain 3(4.9%) each, gastritis and piles 2(3.3%) each while dysentery, purgative and flatulence each were treated with 1(1.6%) of the collected medicinal plants (.

(a) Dominant families in the study area given as a measure of floral diversity, (b) Various gastrointestinal disorders treated with medicinal plants in the study area.

Phytogeographic distribution and utilization status of medicinal plants

The plant collection was carried out in a wide range of altitude ranging from subtropical limatic conditions adjacent to the river Indus to the timber line in upper alpine mountains. Some of the herbaceous plants such as V. serpens and H. telephioides were collected above the timber line. The study areas were designated via an improvised classification into plains (mostly narrow terraces along the river or tributary streams), foothills such as (The Deosai plain) and the mountains (S1 Table). Moreover, the availability of plant species was also recorded as a plant species was flagged as available frequently, occasionally, and rarely. This somehow indicates the conservation status of the plant species from an informant eye.

Methods of preparation

The collected medicinal plants were locally used by various preparation methods. Majority of the plant and plant parts were used in the form of powder contributing 27(34%) followed by direct 19(24%), juice 10(13%), paste 8(10%), roasted form 7(9%), decoction 4(5%), infusion 3(4%) and smoke 1(1%) with least preparation method ( People in the study area directly collect the plant parts and dry either under shade or sun and make powder that is utilized with either water, milk, honey or mixed with any other edible material. The excessive use of powdered material was because of its easy use and storage for longer periods.

(a) Methods of Preparation of folk recipes, (b) Different plant parts used for the treatment of gastrointestinal disorders.

Plant part(s) used for the treatment of gastrointestinal disorders

Different plant part(s) have been used by the locals to treat their GIDs in the study area. Among all, leaves were the dominant parts contributing 25(35%) followed by fruit 18(25), root 11(16%), flower and seed each 4(6%), aerial parts and whole plant 3(4%) and remaining were contributing 1(1%) shown (. Leaves were mostly collected and used by the locals because it is easy to collect the leaves, and these are the most abundantly available parts of the plant. In addition to these leaves are the major photosynthetic machinery which makes almost all the phytochemical in it and due to this, leaves are the most effective plant parts [41].

All the collected plant parts were used orally. Based on the field visits and collection of the specimens, occurrence of the plant species was also mentioned. It was observed that 23(37%) of the medicinal plants were found in mountains followed by 19(31%) in both foothills and plains, 11(18%) species in plains, 4(7%) species in foothills and mountains, 3(5%) were common in all plains, foothills, and mountains while 2(3%) of the plants were restricted to the foothills only (S1 Table). The collected plant species were used in various use forms as 30(48%) of the plant species were used in fresh form followed by dried form 19(31%) and 13(21%) both in fresh and dried form. The collected medicinal plant species were grouped into various life forms as 50(80.6%) were herbs followed by trees 8(12.93%), shrubs 3(4.8%) and climbers 1(1.6%) (S1 Table).

From the collected medicinal plants, ten plants were selected for the preference ranking from ten randomly selected informants. It was observed that H. telephioides (Ledeb.) was the most effective plant to treat GIDs (constipation) and it was assigned with a value 5 by all the key informants and it stood at rank first. Similarly, A. maritima L. was ranked second, Pimpinella diversifolia DC. ranked third and all other plants below this rank (S1 Table). The output of the preference ranking indicated that H. telephioides, A. maritima and P. diversifolia were the most preferable medicinal plants for the treatment of constipation. Direct matrix ranking test was also used to evaluate the functionality of multipurpose uses of the ten randomly selected medicinal plant species mentioned by the five key informants against eight usage categories. The key informants assigned the use values/scores for each plant species (on a scale of 1–5) and categorize each plant. We observed that Prunus persica L. ranked first followed by Ficus carica L., Prunus armeniaca L. each ranked second, and all other plant species ranks were shown in (

The percentage contribution of the use citation of fifteen medicinal plant species representing 50% of use reports/citations was also measured. For this purpose, the use citations of each plant species for various GIDs were summed and its percentage was calculated. It was observed that M. longifolia contributed the highest use citations with 6.4% for overall use citations followed by A. maritima 6.2%, Tanacetum falconeri 5.8%, H. telephioides 4.1%, M. piperita 3.4% while all other plant species contributed below this rang (

List of 15 most cited species representing 50% of use reports/use citations.

Rest of the pie-chart represents 46 plant species.

Fidelity level percentage

Fidelity level for fourteen different GIDs was measured against the collected plant species. It was observed that fidelity level percentage was varied from 100–10 for different plant species. A maximum of 100% fidelity was obtained for six plant species including A. maritima, M. longifolia, M. piperita, A. annua, H. telephioides and Allium cepa against diarrhea and constipation while least fidelity level of 9.8% was shown by Chenopodium album against dysentery ( The highest fidelity level percentage denotes that the plant species is highly preferred for that disease while lower fidelity level indicates that the plant species is not preferred by the informants.

Informant consensus factor

Present study was focused on the medicinal plants used for the treatment of various GIDs. Fourteen different gastrointestinal disorders were cured from 61 collected medicinal plant species by the local community. Among these diseases, the categories with highest ICF value were dysentery, purgative, and flatulence (1.0) each followed by piles, diarrhea, and vomiting (0.98), intestinal worms, constipation, gastritis, stomachic, indigestion and digestive (0.97) while least ICF was calculated for abdominal pain (0.94) (Table 2). The ICF shows that the plant species with highest ICF values were presumed to be more effective and common when used to treat certain disease. Lower ICF values indicated that the informants disagreed on the taxa to be used as a treatment within the disease category. The total use citations for all the plant species that have been used to cure various GIDs was 3248 with highest use citation for stomachic and stomach problems (1150) followed by diarrhea (624) while least use citations were found against flatulence (22) and dysentery (9) (Table 2).

Novelty assessment

The ethnomedicinal data about the GIDs obtained from the study area was compared with the previously published data in Pakistan and neighboring countries to explore the novelty of the plant-based use data of the region. The comparison of the data was broadened with other countries for a much-explored novelty assessment. The comparative analysis identified 19 plant species with specific use reports against different gastrointestinal disorders from the study area for the first time. Among these, T. falconeri, A. maritima & M. piperita were reported to cure vomiting, T. falconeri, R. australe, O. digyna and Viola serpens were identified against stomachic again T. falconeri and V. serpens were reported against diarrhea while H. telephioides, R. australe and O. digyna were identified with novel use to cure constipation. The pharmacological review of plant species was developed with an extensive literature review (Table 3).

Table 3

Novelty assessment of the medicinal plants with novel uses against specific gastrointestinal disorders(bold).

S. No.	Plant name	Local use	Previously reported uses	Reference
1	Artemisia maritima L./RW735	Intestinal worms, diarrhea, vomiting & stomachic	Antiseptic, anthelmintic, antidiabetic, antihypertensive, emmenagogue, antivenom, digestive & cutaneous problems, diarrhea, nausea, fever, Asthma, headache, stomachic, anti-inflammatory, antimalarial, cooling purposes, intestinal worms	[42, 43]
2	Tanacetum falconeri Hook.f./RW739	Diarrhea, vomiting & stomachic	Abdominal problems, asthma, blood pressure & jaundice	[44]
3	Mentha piperita L./RW736	Diarrhea & vomiting	Antidiabetic, antioxidant, hepatoprotective, antipyretic, antispasmodic, analgesic, carminative, diaphoretic, analgesic, anti-diarrhea, anti-microbial, treatment of irritable bowel syndrome (IBS), inflammatory bowel disease, inflammation & dysfunction of the gallbladder & liver diseases	[45–48]
4	Hylotelephium telephioides (Ledeb.) H. Ohba/RW741	Constipation	Skin disease, earache & wounds	[49]
5	Oxyria digyna (L.) Hill/RW740	diarrhea, constipation & stomachic	Appetite disorder, improves digestion	[41, 50]
6	Viola serpens WalL./RW748	diarrhea, constipation & stomachic	Antioxidant, antipyretic, demulcent, diaphoretic, diuretic, asthma, bleeding piles, cancer of throat, cough, fever, skin diseases, headache, emollient, expectorant, febrifuge, purgative, constipation, cough, fever & antinociceptive	[51, 52]
7	Persicaria amplexicaulis (D. Don) Ronse Decr./RW749	Stomach disorders	Sores, wounds, to check excess bleeding during menstruation period, dysentery, cough, rhematic pain, backache, gout, eyesight, purify blood & cause abortion	[53]
8	Saussurea gossypiphora D. Don/RW742	constipation	Asthma, pneumonia, stomach problem, flue, headache, improve circulation, antioxidant, anti-inflammatory and antibacterial, evil spirits, menstrual disorders, hysteria & wound	[54]
9	Aconitum heterophyllum Wall. ex Royle/RW759	Stomachic & intestinal worms	Reduce bile, headache, cold, fever, stomachic, urinary infections, diarrhea, inflammation, antidiarrheal, expectorant, diuretic, hepatoprotective, antipyretic, analgesic, antioxidant, alexipharmic, anodyne, anti-atrabilious, anti-flatulent, anti-periodic, anti-phlegmatic, carminative properties, reproductive disorders, diarrhea, liver, spleen, urinary tract diseases & diabetic	[55]
10	Ribes alpestre Wall.ex Decne./RW764	Ulcer	Antiarthritic, backache, joints pain, jaundice, liver problems, fever, burns, blisters & coolant	[23]
11	Echinops echinatus Roxb./RW770	Abdominal pain	Sexual debility, antimicrobial, analgesic, diuretic, reproductive, hepatoprotective, antioxidant, anti-inflammatory, wound-healing, antipyretic, spermatorrhea, worms, Aphrodisiac, abortifacient, leukorrhea, diabetes, diarrhea, jaundice, hysteria, dyspepsia, hoarseness of throat, cough, asthma, chronic fever, migraine, heart diseases, joint pains, cardiac diseases, lice, ticks, teeth infection, stomachic, analgesic, urinary disorder & hemorrhoids	[56]
12	Rumex hastatus D. Don/RW771	Stomachic & flatulence	Headache, migraine, depression, paralysis, antioxidant, anticholinesterases potentials, laxative, alterative, tonic, rheumatism, skin diseases, piles, bilious complaints, lungs bleeding, blood pressure, tonsillitis, sore throat, anti-poison, digestion, antirusting. flavoring, carminative, diuretic, giddiness & insanity	[57]
13	Pistacia khinjuk Stocks/RW773	Indigestion	Tonic, aphrodisiac, antiseptic, antihypertensive, gastrointestinal, liver, urinary tract, respiratory tract disorders, antioxidant, antimicrobial, antiviral, anticholinesterase, anti-inflammatory, antinociceptive, antidiabetic, antitumor, antihyperlipidemic, antiatherosclerosis, hepatoprotective	[58]
14	Plantago himalaica Pilg./RW779	Stomachic & diarrhea	Dysentery & Stops bleeding from nose	[41, 59]
15	Vitis vinifera L./RW783	Constipation & intestinal worms	Laxative, purgative, diuretic, aphrodisiac, fever, asthma, jaundice, vomiting, stomach problems, piles, joint aches, expectorant, liver tonic, bronchitis, constipation, watering of eyes, lung cancer, burn, boil, wound care, anemia, bronchitis, cold, flu, carminative, costiveness, intestinal spasm, dyspepsia & allergy	[60]
16	Cicer microphyllum Benth./RW786	Indigestion & vomiting	Abscess, swelling of the limbs, poisoning, spleen disorder, colic pain, tonic, mouth & khur disease in goat/sheep	[61]
17	Capparis spinosa L./RW787	Piles & digestive	Toothache, fever, headache, menstruation, rheumatism, convulsion, gout, skin disease, kidney, liver, diabetes, hemorrhoids, ulcers, sciatica, chest disease, febrifuge, dropsy, colds, backache, feminine sterility & dysmenorrheal, anti-dandruff & arthritis	[62, 63]
18	Artemisia annua L./RW793	Diarrhea & vomiting	Antimalarial, anti-inflammatory, anti-microbial, fever, chills, wound healing, intermittent fevers, jaundice, sedative, diarrhea, Anemia, Asthma, eye infections, Cholera, dengue fever, athlete’s foot & eczema, chagas disease, viral hepatitis B & schistosomiasis	[64]
19	Rheum australe D. Don/RW743	stomachic & constipation	Renal function disorders, hyperlipidemia, cancer, digestive problem, diarrhea, laxative, wounds, headache, body pain, joint pain, cure boils, appetizer, astringent, purgative, and health tonic	[65, 66]

Discussion

From the study area, 61 plant species were identified to be used for the GIDs. Among these, maximum number of plants were being used for stomachache followed by diarrhea, constipation, and intestinal worms. Previous studies also confirmed the treatment of GIDs using local medicinal plants [12, 67–70]. The results suggest that locals prefer plant-based remedies for the treatment of GIDs, as in most of the cases herbal preparations provide instant relief without any significant side effect. One of the associated reasons for excessive use of plants for the treatment of GIDs is taboos linked to certain GIDs. For example, people often hesitate to share problems of bad breadth, intestinal gas, and piles comfortably to physicians. Instead, they consult family elders who recommend them to use a locally available medicinal plants or an herbal preparation made up of multiple plants as per their folklore. Other reasons include remoteness of the area and lack of modern health care facilities. It was observed during the survey that there is only one district headquarter hospital (DHQ) situated in Chilas city that is about 23 km away from Buner Das and 38–43 km away from the rest of the study area. In addition, there is no direct road access to the DHQ from the study area instead people travel on foot several miles to get access to the roads. However, there are three dispensaries also exist in the study area that hardly provide primary health care medications for a few commonly occurring ailments.

We calculated FL of each species to quantify relative trust of locals on a particular plant for treating a specific disease. A maximum of 100% fidelity was obtained for six plant species including H. telephioides, A. maritima, M. longifolia, M. piperita, A. annua and Allium cepa against constipation and diarrhea. The plant species having highest FL values indicates a good healing potential against the mentioned disease [71-75]. Based on these findings, we attempted to identify the most preferred plant species with trusted use practices prevailing for treatment of constipation and diarrhea in the study area. H. telephioides for example ranked first for all ten informants selected randomly for preference ranking for the treatment of constipation. Informants consensus factor was also established which indicates the consensus among informants for the treatment of diseases [72]. Among the diseases, highest ICF value was reported for dysentery and flatulence followed by piles, diarrhea, and vomiting and constipation. Higher ICF values indicate consensus in use reports among informants for the treatment of a particular disease. These results suggest that lower intestinal disorders were more commonly prevailed and subsequently the local populace had indigenous healing options in practice.

Owing to poorly explored nature of study area, we hypothesized that these valleys might be hosting some novel medicinal uses of plants. So, we compared our finding systematically with previously published studies in the region at large. It was a pleasant surprise that to best of comparative analysis we carried out, we found 19 medicinal plant species with novel use reports against different GIDs. In our study, for example, fresh leaf juice of A. maritima mixed with mint leaves is taken orally to cure vomiting. The pharmacological review of this plant showed that it has been used as antiseptic, anthelmintic, antidiabetic, antihypertensive, emmenagogue, antivenom, digestive & cutaneous problems, diarrhea, nausea, fever, Asthma, headache, stomachic, anti-inflammatory, antimalarial, cooling purposes and intestinal worms [42, 43].

Fresh leaf juice of T. falconeri grinded with mint leaves is taken orally for the treatment of diarrhea, vomiting and stomachic. The same plant has been reported to be used against abdominal problems, asthma, blood pressure and jaundice [44]. Hylotelephium telephioides is another important plant used to cure against constipation. Half a teaspoon or 5–10 g of the fresh plant part (s) mixed either with water, milk or cured and taken orally. Literature revealed that the plant has been used to cure skin disease, earache, and wounds [49]. Fresh and dried leaves of M. piperita are used as paste, juice and powder mixed with Allium cepa and A. maritima leaves for the treatment of vomiting and also reported previously as antidiabetic, antioxidant, hepatoprotective, antipyretic, antispasmodic, analgesic, carminative, diaphoretic, analgesic, anti-diarrhea, anti-microbial, treatment of irritable bowel syndrome (IBS), inflammatory bowel disease, inflammation and dysfunction of the gall bladder and for liver diseases [45-48]. Dried root powder of R. australe, mixed with butter or milk is taken orally once a day for to cure stomachic and constipation. The same plant was reported from different parts of the world for the treatment of renal function disorders, hyperlipidemia, cancer, digestive problem, diarrhea, laxative, wounds, headache, body pain, joint pain, cure boils, appetizer, astringent, purgative, and health tonic [65, 66]. Fresh leaves of Oxyria digyna, is another important medicinal plant. used to cure diarrhea, constipation and stomachic. the same plant has been reported to be used against appetite disorder and to improves digestion [41, 50]. Present study reported that fresh leaves and fruit of Viola serpens for diarrhea and stomachic. The plant is previously reported as antioxidant, antipyretic, demulcent, diaphoretic, diuretic, cough, asthma, piles, throat cancer, constipation, skin diseases, headache, as emollient, expectorant, febrifuge, purgative, and antinociceptive [51, 52]. Dried root powder of Persicaria amplexicaulis taken orally with butter for stomach disorders in the study area the plant has been reported from the allied valley for the treatment of sores, wounds, dysentery, cough, rhematic pain, backache, gout and to control excess bleeding during menstruation period [53].

The decoction of Saussurea gossypiphora (dried flower) is taken daily before bed and early in the morning to cure constipation. The plant is previously reported to cure asthma, pneumonia, stomach problem, flu, headache, and is used to improve blood circulation, as antioxidant, anti-inflammatory and antibacterial, menstrual disorders, hysteria and wound [54]. Aconitum heterophyllum is used for the treatment of intestinal worms. The plant is reported to treat reproductive disorders, diarrhea, liver, spleen, urinary tract diseases and diabetic, headache, cold, fever, stomachic, urinary infections, diarrhea, inflammation, and as expectorant, diuretic, hepatoprotective, antipyretic, analgesic, antioxidant, alexipharmic, anodyne, anti-atrabilious, anti-flatulent, anti-periodic, anti-phlegmatic, carminative [55].

Another important plant species fresh fruit of Ribes alpestre is taken orally to cure stomach ulcer. The plant is already reported from allied areas as antiarthritic, coolant and to treat backache, joints pain, jaundice, liver problems, fever, burns, blisters and [23]. Fresh leaf infusion of Echinopse chinatus has been used by the locals against abdominal pain. Same plant was reported in other parts of the world against sexual debility and as antimicrobial, analgesic, diuretic, reproductive, hepatoprotective, antioxidant, anti-inflammatory, wound-healing agent, antipyretic, aphrodisiac, abortifacient analgesic, leukorrhea, diabetes, diarrhea, jaundice, hysteria, dyspepsia, hoarseness of throat, cough, asthma, chronic fever, migraine, heart diseases, joint pains, cardiac diseases, teeth infection, stomachic, urinary disorder and hemorrhoids [41, 56]. Fresh aerial parts of Rumex hastatus are collected and grinded with water to obtain juice which is taken orally to cure flatulence and as stomachic. Literature have revealed that, above plant has been used against headache, migraine, depression, paralysis, antioxidant, anticholinesterases potentials, laxative, alterative, tonic, rheumatism, skin diseases, piles, bilious complaints, lungs bleeding, blood pressure, tonsillitis, sore throat, anti-poison, digestion, antirusting. flavoring, carminative, diuretic, giddiness and insanity [57].

Gum of Pistacia khinjuk mixed with honey is taken orally for indigestion. Literature reported that the plant is used as tonic, aphrodisiac, antiseptic, antihypertensive and for GIDs, urinary and respiratory tract infections, as antioxidant, antimicrobial, antiviral, anticholinesterase, anti-inflammatory, antinociceptive, antidiabetic, antitumor, antihyperlipidemic, antiatherosclerosis and hepatoprotective [58].

Local people in the study area use juice and paste of Plantago himalaica leaves against stomachic and diarrhea. The plant has also been reported in literature to be used against dysentery and stops nose bleeding [41, 59]. Fresh leaves and fruit of Vitis vinifera taken orally by locals to cure intestinal worms where, literature review has revealed that the plant has been used as laxative, purgative, diuretic, aphrodisiac, fever, asthma, jaundice, vomiting, stomach problems, piles, joint aches, expectorant, liver tonic, bronchitis, constipation, watering of eyes, lung cancer, burn, boil, wound care, anemia, bronchitis, cold, flu, carminative, costiveness, intestinal spasm, dyspepsia and allergy [60]. Fresh leaves of Cicer microphyllum is used against vomiting and indigestion in the study area while literature have revealed that it is used against abscess, swelling of the limbs, poisoning, spleen disorder, colic pain, tonic, mouth & khur disease in goat/sheep [61]. Shade dried seed powder of Capparis spinosa is mixed with milk or water and taken orally once a week to cure piles and as digestive. Literature reported that the same plant has been used to cure toothache, fever, headache, menstrual cycle disturbance, rheumatism, convulsions, gout, and skin disease. The plant is also reported for kidney problems, liver related ailments, diabetes, hemorrhoids, ulcers, sciatica, dropsy, colds, backache and to cure feminine sterility [62, 63]. Paste of the fresh leaves of Artemisia annua is used orally by the locals to cure serious vomiting while it has been reported in literature for as antimalarial, anti-inflammatory, anti-microbial wound healer and to cure fever, chills, intermittent fevers, jaundice, diarrhea, Anemia, Asthma, eye infections, Cholera, dengue fever, athlete’s foot, eczema, chagas disease, viral hepatitis, schistosomiasis and as sedative [64].

Recently, GIDs have got the attention of scientific community and a new conception of brain-gut-functional axis has emerged to maintain nature’s wellbeing equilibrium [76]. The role of plants and their derivatives is somehow reiterated not only for the treatment of GIDs but also for mitigation of various neurological disorders and psychological condition known to be aggravated by gut discomfort [3, 5, 76, 77]. It is therefore, a need of the hour to extend investigation of aforementioned plants to deduce tangible results for human wellbeing in a holistic manner.

Conclusion

For the first time, this study provided the information on 61 species of traditional applications for the treatment of GIDs by the inhabitants of Diamir, Gilgit, Pakistan. It further enriches our knowledge about the medicinal plant potential and traditional medicinal knowledge of locals about treatment options for GIDs in one of the most far-flung yet iconic region known for its adventurous trekking and mountain climbing. Assessment of conservation status of local flora particularly the plant species with medicinal novelties is highly recommended before any mass collection drive. However, pharmacological evaluation of medicinal flora, with novel therapeutic uses is merited on experimental scale for potential drug leads. Assessment of conservation status of local flora particularly the plant species with medicinal novelties is highly recommended before any mass collection drive. However, pharmacological evaluation of medicinal flora, with novel therapeutic uses is merited on experimental scale for potential drug leads.

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28 Mar 2022

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Reviewer #1: In the present study, the authors have done extensive work in conducting field surveys, and collecting plants and information on traditional use of these plants against gastrointestinal disorders in a remote area of not only Pakistan but possibly also the entire world. The remoteness makes the survey interesting from two points. The first is that new plant species or new uses of already known plant species can be discovered, which has precisely happened in this case. The second is that the remoteness of the area is an impediment to introduction of allopathic medicine, modern hospitals and diagnostic centers; this would suggest that the plants have a possibility of long history of usage and so the medical uses of any plant increase in reliability through their long history of use. Any toxicity or other adverse effects of the plant would have been noted in use over a prolonged time period. On the other hand, the toxicity problem cannot totally be overlooked. Datura stramonium was reported by the authors to be used as a stomachic. The plant, although having analgesic properties, is also very toxic and so exact dose needs to be determined by an experienced practitioner.

Despite the novelties described in the manuscript, several important things are missing, which could have added much necessary information. For instance in Table 3, to assess novelty, nineteen plants have been assessed comparing their use in the survey area versus other reported uses. However, there is no information on the plant part(s) used in the survey area versus other reported areas. Different plant parts (shoots, roots, flowers, seeds) may contain different phytochemicals with different pharmacological properties and medicinal uses. So while the use report may appear to be different in various areas, this may be a consequence of different parts of the plant being used in differing areas.

No detailed information has been given on how the plants were collected; whether they were used in fresh or dried state; if dry then how long can the medicinal activities of a given plant be sustained; preparation of medicine like powder, decoction or other forms, dosage, part of plant used, use of monoherbal or polyherbal formulation; if the same plant is used for more than one gastrointestinal disorder like say diarrhea and constipation did the mode of use differed and how; endangerment of these medicinal plants and any efforts by the locals in their conservation; and the altitude range where the plants can be found.

Gastrointestinal disorders are common in rural areas of practically every developing country. As such, thorough studies of the plants used traditionally to treat such disorders are important, more so because people residing in these areas lack access to modern medical facilities. The authors note that elderly people possessed more knowledge of traditional plant usage. In that case, why were more elderly people not consulted to gather more information? That elderly people know more of their traditional customs is open knowledge; the question is how can the young generation be made more interested in the traditional customs? A short discussion on this topic can also be a valuable addition to the manuscript. An improved version of this manuscript incorporating the changes described above can be of considerable importance to the ethnomedicinal literature.

Reviewer #2: Review report

Dear editor and authors,

The interesting manuscript titled as “Ethnomedicinal appraisal of Plants used for the treatment of gastrointestinal complaints by tribal communities living in Diamir district, Western Himalayas, Pakistan is a novel and significant contribution to science. In this work, Wali and coworkers have performed an extensive ethnobotanical study on the medicinal plants to treat the gastrointestinal complaints by tribal communities living in Diamir district, Western Himalayas, Pakistan. The manuscript is simple, well-organized and easy to read. The theory is also easy to understand and I think it will be read by many researchers. However, before the paper can be considered for publication/acceptance, it is necessary for authors to undertake minor revisions in accordance with the comments of as suggested by me. I hope that this article will appeal wide readership attention and will accelerate progress in this specific field. However, I do find some minor mistakes/shortcomings in the article that the author need to correct before publication. The manuscript is well written but I would recommend revision and improvement of certain sentences. I found this article insightful, applied and quite informative and recommend it for publication with minor revision.

�  The abstract is well-structured, well written and has excellently discussed and concluded the different aspects. However, it would be highly appreciated if authors incorporate sentence about the future perspectives of the research work conducted from present study.

�  Add the word “The” before “resent study was conducted in fairy meadows” in the abstract.

�  There is no future perspective statement in the abstract section. Provide a statement that what next can be done after these ethnobotanical surveys.

�  The introduction section is quite informative and to the point. Authors have comprehensively discussed the notion behind present research work. Authors are advised once again to double check grammar, sentence structure etc, if there any deficiency, fix them accordingly.

�  Provide updated literature to the statement “According to world health organization (WHO) estimates…………. million deaths on 2012 alone. Update 2021-2022 statistics if available.

�  There is no discussion on the overall medicinal plants flora of the said geographical area. Provide few words and discuss about the dominant medicinal plants.

�  What was ecological distribution and phytogeography, utilization status and threats to the surveyed medicinal plants? Do you have some recommendations regarding their conservation aspects?. I think you must add 1-2 lines.

�  Great efforts have been made to write Materials and methods section. All protocols have been explained with full details which is quite fascinating for future researchers to easily pursue their experimental research data collected from field.

�  Results and discussion section is well written, all results have been rightly discussed with relavent data from literature. Add some new 2020-2021 data from literature if available.

�  The conclusion section is comprehensive and well-articulated. Remove results and introductory statements from conclusion section after carefully reviewing manuscript.

�  There is no uniformity in references. Cross check all the references and strictly follow PLOS ONE author guidelines.

Reviewer #3: The manuscript entitled “Ethnomedicinal appraisal of Plants used for the treatment of gastrointestinal complaints by tribal communities living in Diamir district, Western Himalayas, Pakistan”. Authors have analysed and discussed the traditional plant use practices for the treatment of gastrointestinal disorders (GIDs) in Diamir Pakistan Himalayas. They were reported the total of 61 medicinal plant species belonging to 55 genera and 35 families for the treatment of GIDs. Among these, Present study results revealed that 19 out of 61 plant species were documented for the first time with novel medicinal uses to combat GIDs.

The manuscript comprises all the necessary elements of scientific novelty. The study designing and execution of the study were appreciable. I recommend this manuscript for the reconsideration for publication in PlosOne after incorporating minor changes given in below.

COMMENTS FOR THE AUTHOR:

Authors must concentrate on the formatting, and use of symbols, etc., throughout the manuscript.

Continuous line numbers are needed for point out the syntax error, corrections in the manuscript.

Keywords should be unbold.

In introduction, authors have cited the WHO report 2017 it should be referred and cite after 2019 WHO report is better.

Authors should refer few more GIDs articles and include some lines in the introduction section. Because the GIDs information is looks shallow. Better to discuss deeply.

Institutional ethical committee number should be added in the statement of ethics section.

Remove the dot in front of lines in Documentation of Ethnomedicinal Data section.

In sub-title of Results and Discussion section should be “Results”. Because authors have written the separate discussion section.

Framework figure is required. It will be useful to the readers for better understanding of the studied issue.

In materials and method section: authors should explain why each item of methodology was done.

Proper formatting needed in the materials and methods, results, discussion and conclusion section.

In conclusion section authors should provide some future prospectus related to present study.

Reference section should be formatted according to the journal.

Authors should clearly state the figure caption as well as description for ease of understanding of the readers.

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Reviewer #1: Yes: Mohammed Rahmatullah

Reviewer #2: No

Reviewer #3: No

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11 May 2022

Response to the reviewer’s comments

Response to reviewer 1

S. No Reviewer Comment Response

1 R1 Toxicity or other adverse effects of the plant would have been noted

Exact dose of the Datura stramonium needs to be determined. Our survey was somehow targeted to gather plant use information specially for the treatment of GIDs that’s why information on toxicity or adverse effects was not recorded however most of the non-practicing informants and all of the herbal practitioners stressed on use of botanicals in very quantities and this information is well recorded in method of preparation section for example the dose of Datura stramonium “one to two teaspoons of dried seed powder are mixed with 500 ml of water and taken once a week” (Supplementary file 1; row 44, Column M)

2 R1 Need information regarding the use of plant part(s) in the survey area versus other reported areas. We have mentioned the novel use report(s) from the survey area and compared it with already reported plant rather than comparing the plant part(s) used. The literature search works with plant name and hence it was considered less likely that a previous report is overlooked because of different part use information. However, we have carefully rechecked our novelty information and found that novelty of plant species was intact at least up to the part use reported here for a specific disorder

3 R1 No detailed information has been given on how the plants were collected; whether they were used in fresh or dried state; if dry then how long can the medicinal activities of a given plant be sustained. The plants were collected in fresh form initially. Some of the plants were used in fresh form and some in dried form. Information incorporated in supplementary file 1. Mostly locals use the dried plants within 1-2 years of duration.

4 R1 Preparation of medicine like powder, decoction or other forms, dosage, part of plant used, use of monoherbal or polyherbal formulation. Detailed Information regarding preparation method is given in Supplementary file 1 (Columns JKL& M) noteworthy information is also mentioned in figure 3(a). Regarding formulation like monoherbal and polyherbal, information mentioned in supplementary file 1.

5 R1 If the same plant is used for more than one gastrointestinal disorder like say diarrhea and constipation did the mode of use differed and how? Mode of use of same plant for more than one gastrointestinal disorder also mentioned in supplementary file 1. The mode of use is same for more than one disease treated as mentioned in supplementary file 1.

6 R1 Endangerment of these medicinal plants and any efforts by the locals in their conservation. As such locals do not practice any conservation efforts however local people understand that excessive use of plant part(s) can lead the plant for its endangerment, so the locals tend to reduce the overhunting of plant part(s).

7 R1 The altitude range where the plants can be found. The altitude range where the plants were collected is mentioned in Fig 1, which between 35°23'14.3"N latitude and 74°34'44.7"E longitude, at an elevation range between 934-7937 m above the sea level.

8 R1 The authors note that elderly people possessed more knowledge of traditional plant usage. In that case, why were more elderly people not consulted to gather more information? That elderly people know more of their traditional customs is open knowledge; the question is how can the young generation be made more interested in the traditional customs? The population in the study area is not so high and we know that number of elders in a population is always comparatively low. We tried to consult the elders of the survey area aged between 50-60 that comprised up of 27.1% and above 60 years 25.9%. Secondly, in the study area some of the elders don’t want to share their traditional knowledge as they think that their traditional knowledge may be stolen by others. Thirdly, there is health factor of the elders also involved, as the elders are weaker than the youngsters, so they don’t have much energy to discuss with the researcher for a longer discussion.

Response to reviewer 2

1 R2 �  The abstract is well-structured, well written and has excellently discussed and concluded the different aspects. However, it would be highly appreciated if authors incorporate sentence about the future perspectives of the research work conducted from present study. Plant species with maximum medicinal values could be a potential source of novel drug leads to cure gastrointestinal disorders.

2 R2 �  Add the word “The” before “resent study was conducted in fairy meadows” in the abstract. Word “The” added as directed.

3 R2 �  There is no future perspective statement in the abstract section. Provide a statement that what next can be done after these ethnobotanical surveys. Two sentences added at the end of the abstract which are “This showed that occupants of the study area have sound information about ethno-pharmacological consumption of medicinal plants with some novel use reports which may provide the basic data for further pharmacological research. Plant species with maximum medicinal values could be a potential source of novel drug leads to cure gastrointestinal disorders” shows the future perspective statement in the abstract section.

4 R2 �  The introduction section is quite informative and to the point. Authors have comprehensively discussed the notion behind present research work. Authors are advised once again to double check grammar, sentence structure etc, if there any deficiency, fix them accordingly. All done.

5 R2 �  Provide updated literature to the statement “According to world health organization (WHO) estimates…………. million deaths on 2012 alone. Update 2021-2022 statistics if available. Updated as suggested.

6 R2 �  There is no discussion on the overall medicinal plants flora of the said geographical area. Provide few words and discuss about the dominant medicinal plants.

Discussion added as suggested

7 R2 �  What was ecological distribution and phytogeography, utilization status and threats to the surveyed medicinal plants? Do you have some recommendations regarding their conservation aspects? I think you must add 1-2 lines. Added in results a separate subheading as “Phytogeographic distribution and utilization status of medicinal plants” recommendations regarding conservation of medicinal flora added in the conclusion

8 R2 �  Results and discussion section is well written, all results have been rightly discussed with relevant data from literature. Add some new 2020-2021 data from literature if available. Done as suggested

9 R2 �  The conclusion section is comprehensive and well-articulated. Remove results and introductory statements from conclusion section after carefully reviewing manuscript. Conclusion revised according to the suggestions

10 R2 �  There is no uniformity in references. Cross check all the references and strictly follow PLOS ONE author guidelines. All done.

Response to reviewer 3

1 R3 Authors must concentrate on the formatting, and use of symbols, etc., throughout the manuscript. All done.

2 R3 Continuous line numbers are needed for point out the syntax error, corrections in the manuscript. Done

3 R3 Keywords should be unbold. Done.

4 R3 In introduction, authors have cited the WHO report 2017 it should be referred and cite after 2019 WHO report is better. Done as suggested

5 R3 Authors should refer few more GIDs articles and include some lines in the introduction section. Because the GIDs information is looks shallow. Better to discuss deeply. Updated as suggested

6 R3 Institutional ethical committee number should be added in the statement of ethics section. Ethical committee number added as UPR/HACE/01/13/21. Dated March 16, 2021.

7 R3 Remove the dot in front of lines in Documentation of Ethnomedicinal Data section. Done

8 R3 In sub-title of Results and Discussion section should be “Results”. Because authors have written the separate discussion section. Done

9 R3 Framework figure is required. It will be useful to the readers for better understanding of the studied issue. Framework figure added with the map of the study area (Fig.1). As there is a limit of 5 figures from PLOS at max.

10 R3 In materials and method section: authors should explain why each item of methodology was done. Explained

11 R3 Proper formatting needed in the materials and methods, results, discussion, and conclusion section. Done.

12 R3 In conclusion section authors should provide some future prospectus related to present study. Done as suggested

13 R3 Reference section should be formatted according to the journal. All done.

14 R3 Authors should clearly state the figure caption as well as description for ease of understanding of the readers. All done.

Submitted filename: Response to Reviewer.docx

Click here for additional data file.

23 May 2022

Ethnomedicinal appraisal of Plants used for the treatment of gastrointestinal complaints by tribal communities living in Diamir district, Western Himalayas, Pakistan

PONE-D-22-02797R1

Dear Dr. Khan,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Jen-Tsung Chen, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

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The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

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PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Authors have answered all my previous comments.

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Reviewer #1: Yes: Mohammed Rahmatullah

30 May 2022

PONE-D-22-02797R1

Ethnomedicinal appraisal of Plants used for the treatment of gastrointestinal complaints by tribal communities living in Diamir district, Western Himalayas, Pakistan

Dear Dr. Khan:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

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on behalf of

Dr. Jen-Tsung Chen

Academic Editor

PLOS ONE