In Vivo Anti-inflammatory, Analgesic, and Sedative Studies of the Extract and Naphthoquinone Isolated from Diospyros kaki (Persimmon).

Anti-inflammatory, analgesic, and sedative medicine is used with numerous side effects, including peptic ulcer, headache, addiction, and other complications. In this regard, discovery research is undergoing a process to discover effective, safe, and economical drugs with no side effects. The aim of this study was to assess chloroform extracts and isolated compounds for anti-inflammatory, analgesic, and sedative activities in animal models. The anti-inflammatory potential was measured by using the carrageenan-induced and histamine-induced paw edema procedure, while the analgesic potential was determined using a hot plate analgesiometer. The sedative effect was observed in an animal model for screening of the locomotor effect of the extract and isolated compound 1. Our data exhibited that the extract and compound 1 attenuated carrageenan-induced and histamine-induced paw edema (93.98 and 89.54%, respectively). Furthermore, compound 1 attenuated biphasic edema associated with histamine and prostaglandins. The chloroform extract showed a moderate analgesic effect; however, compound 1 showed a significant analgesic potential (p < 0.001) by increasing the latency time of the animals in the thermally induced algesia model. Compound 1 exhibited a significant sedative effect and dose-dependent analgesic activity. It is concluded that the chloroform extract and compound 1 showed remarkable anti-inflammatory, analgesic, and sedative activities. This research work strongly rationalizes the folkloric usage of Diospyros kaki in the treatment of inflammation, pain, and insomnia.

Introduction

Medicinal plants are the factories of phytochemicals that produce different bioactive compounds in the most selective and precise way.[1] Plants produce both primary and secondary metabolites. Plants use primary metabolites for basic life functions like growth, cell division, storage, respiration, and reproduction; these include the compounds involved in the Krebs cycle, glycolysis, photosynthesis, etc. Secondary metabolites are also known as secondary products, natural products, phytochemicals, or specialized metabolites, which are not directly involved in the growth, development, and reproduction of the plants.[2] The medicinal effect of plants is oriented toward secondary metabolites. Phytochemical composition is a key in the usage of medicinal plants for the treatment of various diseases in traditional systems. In the modern medicinal system, natural products provide lead compounds for the production of new drugs in the pharmaceutical industry.[1−3] There are many secondary metabolites that were investigated and used as major constituents in modern medicine. Various plants are investigated for the discovery of medicine because plant-based drugs are less toxic and more effective.

Diospyros kaki belongs to the family Ebenaceae, which is commonly known as oriental persimmon or Japanese persimmon.[4] It is a deciduous tree having a round scattering crown that grows up to 20–30 feet tall. It is distributed in Pakistan, Burma, China, and Korea, while it is widely cultivated in Japan.[5]D. kaki is a medicinally important plant used in various traditional systems for the treatment of various diseases. Traditionally, D. kaki is used for heart problems like the treatment for stroke, angina, and also internal hemorrhage. It is also used for the cure of constipation, diarrhea, and burns. In the literature, it is documented as treatment for bronchial problems like cough and apoplexy. It has the potency to overcome skin problems, including pimple skin eruption and many other skin diseases.[6−8] Plant extracts are documented as sedative, antioxidant, antimutagenic, anti-allergic as well as antihistaminic, and antihypertensive. In the literature, various parts of D. kaki have been reported as antidiabetic and anti-inflammatory.[7,8] Phytochemical D. kaki is enriched in various secondary metabolites comprising vitamins, flavonoids, terpenoids, lipids, sugars, vitamins, and tannins.[9] The root extract has been documented for its various secondary metabolites, including 2-bromo-1,4-naphthoquinone, 5-hydroxy-2-methyl-1,4-naphthoquinone, 5-hydroxy-1,4-naphthoquinone, and 2-hydroxy-1,4-naphthoquinone.[10] Similarly, carotenes, resins, chlorophyll, polysaccharides, kryptoxanthin, hemicelluloses, amino acids, and lignin are also reported from leaves of D. kaki.(11) Based on phytochemistry and pharmacological application, we selected this plant to explore its hidden properties for the treatment of different diseases. The main aim of this study was to screen the extract and naphthoquinone (1) of D. kaki for in vivo anti-inflammatory, analgesic, and sedative activities.

Results

Anti-inflammatory Effect

Our results showed that the defatted chloroform extract displayed a good effect at a higher dose (200 mg/kg) in the carrageenan-induced paw edema model. At the initial inflammatory phase, 30.98% (1st hour) and 45.09% (2nd hour) attenuation in edema was noted, which was improved with the passage of time. The maximum effect (73.87%) was observed after the 3rd hour of the experimental duration, while 52.98 and 45.97% were observed after the 4th and 5th hour of the experimental duration. Similarly, the anti-inflammatory responses of the tested compound 1 were 50.90 and 75.09% in the initial phase (1st and 2nd hour) and a maximum of 93.98% in the second phase (3rd hour) of the experiment as shown in Figure , while responses were 76.98 and 70.76% after the 4th and 5th hour of the experimental duration.

Figure 1

Anti-inflammatory activity of chloroform fraction (200 mg/kg) and compound 1 (15 mg/kg) purified from D. kaki on carrageenan-induced paw edema in mice. All values are expressed as mean ± SEM for all groups of animals (n = 6).

Similarly, in the case of the histamine-induced paw edema model, the defatted chloroform extract exercised excellent reduction in paw edema for the initial phase of the experiment (50.32%) at the 1st hour and a significant effect (61.22%) after the 2nd hour while a good effect (54.87) after the 3rd hour and 45.86, 40.66, and 38.87% after the 4th, 5th, and 6th hour of the experimental duration. A similar significant effect (89.54 and 93.87%) was observed in the compound 1-treated group after the 1st and 3rd hour while 86.87% in the 3rd hour and 74.78, 63.66, and 53.76% after the 4th, 5th, and 6th hour as displayed in Figure .

Figure 2

Anti-inflammatory activity of chloroform fraction (200 mg/kg) and compound 1 (15 mg/kg) purified from D. kaki on histamine-induced paw edema in mice. All values are expressed as mean ± SEM for all groups of animals (n = 6).

Analgesic Effect

The results of the analgesic effect of the chloroform extract and compound 1 are displayed in Table . An excellent (p < 0.001) analgesic activity was observed at various doses of the extract (25, 50, 100, and 200 mg/kg) and pure compound 1 (2.5, 5, 10, and 15 mg/kg). Compound 1 increased the latency time from the start of the experiment and maintained significant increases (p < 0.001) up to 2 h. Meanwhile, no significant activity was noticed at lower doses. The extract showed a low pain-relieving effect as compared to compound 1.

Table 1

Analgesic Effect of Chloroform Extracts and Compound 1 Isolated from D. kakia

		time (min)
group	dose (mg/kg)	30	60	90	120
saline	10 mL/kg	9.23 ± 0.10	9.21 ± 0.9	9.22 ± 0.12	9.19 ± 0.08
tramadol	5	25.01 ± 0.09***	27.17 ± 0.91***	25.82 ± 0.78***	25.18 ± 0.49***
chloroform	25	12.59 ± 0.44	14.88 ± 0.40	15.12 ± 0.85	14.24 ± 0.80
	50	13.00 ± 0.58	15.08 ± 0.42	14.60 ± 0.64	13.68 ± 0.87
	100	14.08 ± 0.65*	16.19 ± 0.50*	15.98 ± 0.76*	14.88 ± 0.97*
	200	15.12 ± 0.29**	17.19 ± 0.54**	16.85 ± 0.87**	15.78 ± 0.76**
compound 1	2.5	19.13 ± 0.46**	20.48 ± 0.50**	19.90 ± 0.58**	19.68 ± 1.52**
	5	20.86 ± 0.58**	22.01 ± 0.66**	21.50 ± 0.78**	21.41 ± 1.08**
	10	21.52 ± 0.63***	23.12 ± 0.77***	22.15 ± 0.83***	21.99 ± 1.14***
	15	22.53 ± 0.80***	24.12 ± 0.93***	23.30 ± 0.90***	23.03 ± 1.32***

The data are expressed in the table as the mean ± standard error of the mean (SEM) of latency time in seconds for each group (n = 6); also, statistical analysis of variance (one-way) was performed followed by multiple comparison through Dunnett’s post hoc test, where *p < 0.05, **p < 0.01, and ***p < 0.001.

Sedative Effect

The results related to the sedative potential of the chloroform extract and purified compound 1 are displayed in Table . The extract exhibited low sedative activity at tested doses (25, 50, 100, and 200 mg/kg). Compound 1 exhibited significant sedation (p < 0.001) at 15 mg/kg by hindering the mobility of animals in a special box.

Table 2

Sedative Effect of Chloroform Extracts and Compound 1 Isolated from D. kakia

treatment	dose (mg/kg)	line crossed
diazepam	0.5	6.87 ± 0.12***
chloroform extract	25	100.26 ± 2.01
	50	95.87 ± 2.08
	100	91.22 ± 2.07
	200	82.87 ± 1.88
compound 1	2.5	72.98 ± 1.80*
	5	65.09 ± 1.60*
	10	53.98 ± 1.30**
	15	42.98 ± 1.18***

The data are expressed in the table as the mean ± standard error of the mean (SEM) of the number of lines crossed for each group (n = 6); also, statistical analysis of variance (one-way) was performed followed by multiple comparisons through Dunnett’s post hoc test, where *p < 0.05, **p < 0.01, and ***p < 0.001.

Discussion

D. kaki is an indigenous medicinal plant having multitraditional uses, such as treatment for internal hemorrhage and stroke, a sleep inducer, treatment for pain and inflammation, an analgesic, and cure for constipation, diarrhea, and burns.[7,8,10] The extract of D. kaki has been documented with sedative, antioxidant, antimutagenic, anti-allergic as well as antihistaminic, and hypertensive activities. Different parts of D. kaki have been reported to have antidiabetic,[10] anti-inflammatory,[12] antioxidant,[13] anti-human immunodeficiency virus (HIV), and anticancer properties.[14]

The bioactivities of the plant extract are due to the bioactive phytochemicals in the extract. Therefore, the isolation of these phytochemicals followed by biological screening is essential to find the purified molecules for their biological potency. In the present study, the extract and its isolated molecules have been assessed for anti-inflammatory, analgesic, and sedative effects.

Previously, naphthoquinones and their derivatives have been reported for their anti-inflammatory, analgesic, and sedative potencies.[15−17] Compound 1 isolated from D. kaki (persimmon) showed anti-inflammatory, analgesic, and sedative activities and attenuated inflammation in animal models like histamine-induced and carrageenan-induced paw edema models. Carrageenan causes inflammation in two phases:[18,19] the initial phase edema is attributed to the localization of histamine, bradykinins, and serotonin, while the other phase is due to overproduction of prostaglandins (PGs). Our results showed that the chloroform extract exhibited a moderate effect at a higher dose, while compound 1 inhibited biphasic edema by carrageenan, which is an indication that the pure compound 1 is a histamine and PG blocker.

Likewise, the naphthoquinone class of phytochemicals has been documented with antihistaminic potentials.[20] The antihistaminic-type potential may be similar to an H1 receptor blocker (pheniramine), which is a sedative.[21] Our data exhibited that the tested extract and compound 1 are good sedatives. PGs are the product of arachidonic acid via the cyclooxygenase (COX) pathway. Most of the COX inhibitors are painkillers and anti-inflammatory drugs with various gastric and renal side effects. That is why the discovery of new, novel, effective, and safe COX inhibitors is focused on by the researchers across the globe. In the thermally induced pain model, the extract and compound 1 showed significant activity. The promising anti-inflammatory, analgesic, and sedative potentials of compound 1 isolated from the chloroform fraction provided a strong scientific explanation for the folkloric usage of D. kaki in the cure of inflammation and pain conditions.

Materials and Methods

Plant Collection

The plant sample of D. kaki was collected from Lower Dir, Toormang, KPK, Pakistan, in December 2018. The plant specimen was identified by Dr. Muhammad Ilyas, Department of Botany, University of Swabi, KPK, Pakistan. Voucher specimen no. UOS/Bot 43 was placed in a botanical garden of the Department of Botany, University of Swabi, Pakistan.

Extraction and Isolation

The plant roots (5 kg) were dried in air for 18 days and then subjected to a grinder to obtain a fine powder. The fine powder was subjected to cold extraction by using chloroform for 14 days as per standard methods.[22] The reddish extract obtained was concentrated with the help of a rotary evaporator at low temperature and reduced pressure to collect the crude extract (56.12 g). The chloroform extract was defatted with hexane using a Soxhlet apparatus to obtain the chloroform extract (48.87 g). The chloroform fraction (14 g) was assessed by normal-phase column chromatography (CC) using column silica gel. The elution of the column was done by serially increasing the polarity of the solvent system comprised of n-hexane (nonpolar) and ethyl acetate (polar) (the ratio of n-hexane:ethyl acetate was serially increased from 100:0 to 40:60), which resulted in isolation of 17 fractions (DK-1 to DK-17). All obtained fractions from column chromatography were subjected to TLC profiling. The DK-8 fraction was subjected to repeated normal-phase CC by using n-hexane and ethyl acetate (88:13), which yielded red needle-like crystals. The obtained crystal was washed with hexane and acetone, which gives compound 1. The chemical structure of dinaphthodiospyrol S, 1′,4′-dihydroxy-1,4-dimethoxy-3,7′-dimethyl-[2,2′-binaphthalene]-5,5′,8,8′-tetraone (1; Figure ), was identified by comparing the physical and spectroscopic data with our recently published data.[11]

Figure 3

Chemical structure of dinaphthodiospyrol H (1).

Animals

Fit BALB/c mice weighing 22–27 g, which were obtained from the National Institute of Health Islamabad Pakistan, were used in the current biological screening. All animals were placed under standard laboratory conditions and fed with standard water and diet. The design of study was approved by the Ethical Committee of the University of Swabi, KPK, Pakistan.

Anti-inflammatory Activity

The anti-inflammatory response of the defatted chloroform extract and compound 1 was observed with carrageen-induced paw edema and histamine-induced paw edema models as per the recently reported protocol.[23] The BALB/c mice were distributed into different groups of six mice each. These divided groups of animals comprise a positive control (diclofenac and loratadine; 5 mg/kg) and a negative control (10 mL/saline). Tested groups of animals were administrated with defatted chloroform extract (25, 50, 100, and 200 mg/kg) and pure compound 1 (2.5, 5, 10, and 15 mg/kg). After 30 min of the intraperitoneal treatment, 0.1 mL of histamine and 1% carrageen (0.05 mL) were transdermally injected into the right paw of each animal. The paw edema was monitored in each animal for several hours (1–6 h) after histamine and carrageenan injection by using a plethysmometer. Thus, the anti-inflammatory response of the defatted chloroform extract and pure compound 1 against paw edema was calculated using the following formula:where A represents paw edema of the control, and B represents paw edema of the tested extract/compound.

Analgesic Activity

The defatted chloroform extract and purified compound 1 were screened for analgesic effect as per the reported method.[23,24] Based on the shape and pattern, animals were divided into various groups of six. The first group was treated with normal saline (10 mL/kg) as a control for statistical analysis, while the second group was treated with a standard drug (tramadol; 5 mg/kg). All groups of mice were treated with defatted chloroform extract (25, 50, 100, and 200 mg/kg) and pure compound 1 (2.5, 5, 10, and 15 mg/kg). The analgesic activity was determined with the help of a hot plate analgesiometer as per the standard procedure. After 30 min of sample treatment, each group of animals was tested for analgesic potency on the hot plate, and the time of latency was recorded in seconds. The percent analgesic potential of the defatted chloroform extract and purified compound 1 was determined as per the reported method.

Sedative Activity

All animals were grouped as per the above method apart from the control, which was administrated with 0.5 mg/kg diazepam. The bioactivity was determined as per our previously published method.[23,25,26] As per this method, after 30 min of treatment, each group of mice was placed in a specially designed box, and the lines crossed by each mouse were noted. In each group, those animals with the delayed movement were considered as sedative, while animals that crossed more lines were reflected as nonsedative.

Statistical Analysis

The obtained results of in vivo biological screening are shown as the mean ± standard error of the mean (SEM) to find the significant difference (p = 0.05 or 0.01) in the tested groups of animals. All the obtained results were assessed by one-way analysis of variance (ANOVA). Statistical analysis was done with the help of Dunnett’s multiple assessment screening using GraphPad Prism 5, with significant difference at p ≤ 0.05.

Conclusions

D. kaki is traditionally used for inflammation, constipation, and diarrhea and as a sedative and a painkiller. Results obtained in the present study exhibited that the chloroform extract and compound 1 showed excellent anti-inflammatory, analgesic, and sedative effects. Thus, our investigation provided a scientific rationale to the traditional usage of the title plant for the treatment of various diseases. Furthermore, the isolated compound 1 should be a new candidate for further detailed assessment and clinical investigations. Therefore, more detailed studies are required to establish the safety and effectiveness of compound 1 and the chloroform extract.