Evaluation of In vivo Antimalarial Property of Nyctanthes arbor-tristis (Night Jasmine) Leaves.

BACKGROUND: Nyctanthes arbor-tristis (NAT) is an ornamental garden plant traditionally used for treating many diseases such as helminthiasis, arthritis, and malaria. AIMS: The aim of this study was to validate the ethnobotanical uses of the antimalarial activity of leaves of NAT by in vivo tests.
MATERIALS AND METHODS: Leaves of NAT were identified and authenticated and phytoconstituents of NAT were identified. The antimalarial activity of NAT was studied in in vivo for its schizonticidal activity, repository activity, and curative tests in Swiss albino mice by using Plasmodium berghei (ANKA). STATISTICAL ANALYSIS USED: One-way ANOVA was done for comparison of different groups followed by post hoc analysis (Tukey-Kramer multiple comparison tests). Level of significance was at P < 0.05.
RESULTS: The mean schizonticidal activity of NAT increased from 14.21 to 46.15 (P < 0.01) with doses ranging from 100 to 200 mg/kg compared to 67.29 with that of chloroquine (CQ). The repository activity with NAT doses 100-200 mg/kg increased from 12.91 to 42.85 (P < 0.01) compared to 78.79 in pyrimethamine 1.2 mg/kg/day. In Rane's test, there was chemosuppression in range of 55.50-65.02 (P < 0.01) with NAT in doses of 100-200 mg/kg compared to 74.15 with that of CQ 5 mg/kg.
CONCLUSIONS: The antiplasmodial activity of NAT might be like that of artemisinin by producing oxidative stress mostly due to the iridoid glycosides. The active phytoconstituent(s) responsible may be tested individually or in combination both by in vitro and in vivo studies to identify the active chemical ingredient. Copyright:

INTRODUCTION

Malaria caused by six species of malaria parasites is a global health problem. According to the World Malaria Report 2017 (WHO), around 216 million cases and 445,000 deaths were reported in the year 2016.[1] The Plasmodium falciparum malaria occurs mostly in sub-Saharan Africa and South East Asia.[23]

Cinchona bark (sweet bark or Peruvian bark) has been used by indigenous Peruvians to treat shivering and fever. By 1640, bark of cinchona was used in Europe as powder and given as beverages to cure “fevers and tertians.” The bark contains quinine which is used for the treatment of malaria.[4] Chinese herbal drug “qinghao” from Artemisia annua has been used by the Chinese for malaria for more than 2000 years. In 1972, Chinese scientist extracted artemisinin, a sesquiterpene lactone the major antimalarial ingredient.[5]

Night jasmine or Nyctanthes arbor-tristis (NAT) described as “a sacred plant” is found in sub-Himalayan region in India and often used as an ornamental and medicinal plant. The plant belongs to Oleaceae family.[6] It has an average life span of 10 years and grows up to height of 3–4 m [Figure 1]. Leaves of the plant are hairy green and rough with serrated margins, oval apex and measure about 5–10 cm long and 2.5–5 cm wide. The plant known as Parijat in Sanskrit, is used in various traditional systems of medicine in for a wide range of diseases like arthritis and for worm infestations. [7] Antimicrobial, antihelminthic, anti-inflammatory, antioxidant, immunomodulatory, and anticancer properties of the plant have been studied.[8910111213] The plant (NAT) has been quoted in the classical texts of Chakradutta for “Visham Jwara” (malaria).[14]

Figure 1

Nyctanthes arbor-tristis plant with stem and leaves

The antimalarial potential of the Night Jasmine plant leaves (NAT) has been explored by reverse pharmacology approach.[1516] In this study, we made an attempt to isolate phytochemical constituents and elucidate mechanisms of pharmacological action on the basis of their therapeutic applications, especially in malaria.

Objectives of the study

The objective of this study was to identify the phytoconstituents of night jasmine (NAT) and gather evidence (by in vivo test).

MATERIALS AND METHODS

Approval was obtained from the institutional animal ethics committee. The principles of good laboratory practice and CPCSEA guidelines were followed. Fresh leaves of NAT were collected, identified, and authenticated at Regional Plant Research Centre, Bhubaneswar, India. After cleaning and drying under shade, dry powder of leaves was made and weighed and extraction was done in 95% ethanol. Phytochemical screening was done by column chromatography method.[17]

Healthy male Swiss albino mice (BALB/c) of 20–25 g were used. The animals were provided standard diet and water was provided ad libitum and were provided 12 h light and 12 h dark cycles with adequate ventilation. For acute toxicity test, the mice were divided into five groups of ten mice each. They were given the drug extract NAT intraperitoneally in doses of 100, 200, 300, 400, and 500 mg/kg. Determination of LD50 was done by graphic method of Miller and Tainter (1944).[18] Two doses such as 10% and 20% of LD50 of NAT were taken, and one intermediate dose was chosen.

Evaluation of the schizonticidal activity on early infection (4-day test) was done with thirty albino mice randomly divided into five groups of six each.[19] The chloroquine (CQ)-sensitive Plasmodium berghei (ANKA) strain was obtained from National Institute of Malaria Research, Dwarka, Delhi. The inoculums consisted of 0.2 ml of 1 × 107 of parasitized RBCs. The NAT extract was administered as 100, 150, and 200 mg/kg/day. CQ 5 mg/kg/day served as positive control and normal saline 0.2 ml as control. After 4 days, the tail vein blood was collected. Thin blood films were stained with Giemsa stain. Mean parasite count per 500 erythrocytes in a random field was estimated. The mean percentage inhibition of parasitemia was calculated as:

For evaluation of the repository activity, five groups of six animals were administered 100, 150, and 200 mg/kg/day doses of NAT, 1.2 mg/kg/day pyrimethamine (standard group), and distilled water (control) for 4 consecutive days.[20] On day 5, the mice were inoculated with P. berghei. After 72 h of drug administration blood smears were prepared from tail vein of the animals and parasitemia level was estimated.

Evaluation of the schizonticidal activity on established infection was done with n = 30 mice. On day 0, standard inocula of 1 × 107 infected erythrocytes were injected in mice intraperitoneally.[21] After 72 h, the mice were divided into five groups (n = 6). NAT extract (100, 150, and 200 mg/kg/day) doses were orally administered. Chloroquine (CQ) 5 mg/kg/day was given to the positive control group and an equal volume of distilled water was given to the negative control group. The drug and extract were given once daily at the same time for 5 days. Thin films were made from tail vein blood daily for 5 days to estimate the parasitemia level.[21]

In all the tests, the mean survival time (MST) of the mice was monitored in each group for 30 days.

Data are expressed as mean ± standard deviation. One-way ANOVA was done for comparison of data of different groups followed by post hoc analysis (Tukey-Kramer multiple comparison tests). Level of significance was at P < 0.05.

RESULTS

Phytochemical screening of the ethanolic leaf extract of NAT revealed the presence of compounds such as saponins, alkaloids, terpenes, flavonoids, and glycosides by column chromatography method [Figure 3].

Figure 3

Graphical abstract

Structural similarity between (a and b) iridoid glycosides from Nyctanthes arbor-tristis and artemisinin derivatives

In Acute Toxicity test the median lethal dose (LD50) of NAT extract was found to be 977.23 mg/kg [Table 1]. Results of the Acute Toxicity test for Nyctanthes arbortristis leaf extract for determination of LD50 (median lethal dose) after intraperitoneal injection in Swiss albino mice (n+10).

Table 1

Results of the lethal doses of Nyctanthes arbor-tristis for the determination of lethal dose 50 value after intraperitoneal injection in Swiss albino mice (n=10)

Group	Dose (mg/kg)	Log dose	Dead (%)	Probit
1	100	2	10	3.77
2	200	2.30	15	4.05
3	300	2.47	21	4.19
4	400	2.60	28	4.42
5	500	2.69	42	4.8

LD50 of NAT extract as calculated by regression analysis method, LD50=977.23 (mg/kg). LD: Lethal dose, NAT: Nyctanthes arbor-tristis

On evaluation of the schizonticidal activity in 4 Day Test we found that the ethanolic leaf extract of NAT produced significant dose dependent schizonticidal activity. The mean percentage inhibition of maturation to schizonts ranged from 14.21 to 46.15 with NAT doses 100-200 mg/kg body weight compared to 67.29 with that of chloroquine (standard positive control). The mean survival time of the animals also significantly increased ranging from 14.75 to 16.83 days compared to 17.67 days with that of chloroquine [Table 2].

Table 2

Blood schizonticidal activity of ethanolic leaf extract of Nyctanthes arbor-tristis during early Plasmodium berghei infection (4-day test) in Swiss albino mice (n=6)

Drug	Dose (mg/kg/day)	Mean parasitemia (mean±SD)	Mean percentage inhibition	MST in days
NS	0.2 ml	43.33±0.82		12.00±0.63
NAT1	100	37.17±0.75b	14.21	14.75±0.50a
NAT2	150	32.50±0.83c	24.99	16.17±0.75b
NAT3	200	23.33±0.81c	46.15	16.83±0.41c
CQ	5	14.17±1.17c	67.29	17.67±0.52c

Values expressed as mean±SD. P<0.001 (by one-way ANOVA). Significance relative to control/NS group. aP<0.05, bP<0.01, cP<0.001. SD: Standard deviation, NS: Normal saline, NAT: Nyctanthes arbor-tristis, CQ: Chloroquine, MST: Mean survival time

The ethanolic NAT extract also exhibited dose dependent schizonticidal activity on evaluation of Repository Activity of the drugs. The mean percentage inhibition of parasitemia ranged from 12.91 to 42.85 in the NAT groups with doses of 100-200 mg/kg body weight of NAT extract compared to 78.79 in Pyrimethamine group. The mean survival time ranged from 14.25 to 16.17 days in NAT group compared to 17.50 days in Pyrimethamine group [Table 3].

Table 3

Repository activity of Nyctanthes arbor-tristis extracts against Plasmodium berghei infection in Swiss albino mice (n=6)

Drug	Dose (mg/kg/day)	Mean parasitemia (mean±SD)	Mean percentage inhibition	MST in days
NS	0.2 ml	36.17±0.75		11.67±0.52
NAT1	100	31.5±0.55b	12.91	14.25±0.50b
NAT2	150	27.17±0.98c	24.88	14.67±0.52c
NAT3	200	20.67±0.81c	42.85	16.17±0.41c
Pyrimethamine	1.2	7.67±0.82c	78.79	17.50±0.55c

Values expressed as mean±SD. P<0.001 (by one-way ANOVA). Significance relative to control/NS group. aP<0.01, bP<0.001. SD: Standard deviation, NS: Normal saline, NAT: Nyctanthes arbor-tristis, MST: Mean survival time

Rane's Test was done on established P. berghei infection. The NAT extract in doses of 100-200 mg/kg body weight showed significant chemo suppression in range of 55.50 to 65.02 compared to 74.15 with that of CQ group. The mean survival time of the animals in the NAT group was 13.25 to 15.5 days compared to 16.67 days in CQ group [Table 4].

Table 4

Antiplasmodial activity of leaf extract of Nyctanthes arbor-tristis in mice with established infection of Plasmodium berghei (curative test/Rane’s test) (n=6)

Drug	Dose (mg/kg/day)	Percent of parasitemia (mean±SD)				Mean percentage inhibition	MST (days)
		Day 3	Day 4	Day 5	Day 7
NS	0.2 ml	32.17±0.75	35.83±0.75	41.83±0.75	43.83±0.83		11.67±0.52
NAT1	100	29.33±0.82	27.50±0.55b	24.17±0.89c	19.50±0.84c	55.50	13.25±0.50a
NAT2	150	28.33±0.52a	25.33±0.52c	22.17±0.75c	17.83±0.75c	59.32	14.50±0.55c
NAT3	200	26.67±0.82b	23.17±0.75c	20.67±1.03c	15.33±0.82c	65.02	15.50±0.54c
CQ	5	25.33±0.52c	21.50±0.55c	17.50±0.82c	11.33±1.21c	74.15	16.67±0.52c

Values expressed as mean±SD. Significance relative to control/NS group. aP<0.05, bP<0.01, cP<0.001. SD: Standard deviation, NS: Normal saline, NAT: Nyctanthes arbor-tristis, CQ: Chloroquine, MST: Mean survival time

DISCUSSION

There was dose-dependent inhibition of parasitemia compared to the CQ in 4-day test. The NAT extract showed repository activity in dose-dependent manner and comparable to pyrimethamine. On established P. berghei infection by Rane's test, NAT extract showed significant dose-dependent chemosuppression comparable to that of CQ. There was an increase in the MST of the animals in all the tests. The results from our study are in sync with the previous in vivo studies.[2223]

By column chromatography method, the extract yielded alkaloids, flavonoids, glycosides, and terpenes. Various studies and reviews indicated that the iridoid glycosides may be responsible for the antiparasitic activity. In leishmaniasis, NAT exerts its action by altering the redox potential.[10] It is postulated that like artemisinin, the iridoid glycosides exert their action by producing oxidative stress.

In addition, the seeds of the plant also yield arbortristoside which may exhibit anticancer effect by immunomodulation and by induction of cytokines.[13] Experimentally, it has been shown that artemisinin-based combination therapy can be achieved by using NAT extract.[23] The iridoid glycosides also have anti-inflammatory action, and they have been reported to inhibit tumor necrosis factor-α, interleukin (IL)-6, and IL-10.[912] This action on platelets also prevents platelet aggregation.[16] Some of the plant products like Picrorhiza kurroa (Katurohini/Kutki) which contains iridoid glycosides (Kutkin) have been well documented in ancient scripture “Caraka Samhita.”[14] The immunomodulatory, antioxidant, and anticancer properties of the different parts of the plant have been attributed to the presence of iridoid glycosides.[912]

The limitations of the study are that the active phytoconstituent(s) responsible have not been tested individually or in combination both by in vitro and in vivo studies. This might help for development of a novel antimalarial drug from NAT for malaria eradication.

CONCLUSIONS

The leaves of night jasmine plant (NAT) used in traditional medicine for Visham Jwara (malaria) were identified. It was observed that NAT extract exhibited dose-dependent gradual improvement in clearing parasitemia and increased the survival time in mice. Among the chemical constituents, it was identified that the iridoid glycosides may be playing a vital role in the antimalarial activity.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.