Impact factor: 0.3397/ICV: 4.10 ISSN: 0976-7908 19

Pharma Science Monitor 11(1), Jan-Mar 2020

PHARMA SCIENCE MONITOR AN INTERNATIONAL JOURNAL OF PHARMACEUTICAL SCIENCES

Journal home page: http://www.pharmasm.com

ALSTONIA SCHOLARIS: A POTENT ASIAN MEDICINAL (A COMPREHENSIVE PHYTOPHARMACOLOGICAL REVIEW) Mittal Rudani, Yogesh Chhotala, Rakesh Prajapati*, Girish Sailor, Vineet Jain Department of Pharmacognosy, Bhagwan Mahavir College of Pharmacy, Gujarat Technological University, Surat, Gujarat, 395017, India.

ABSTRACT Alsotnia scholaris (Family: / Araceae) (AS) is an annual herbaceous plant with a long history of traditional medicinal uses in many countries in the world, especially in the tropical and subtropical regions. Since, there are few articles on this plant, the present review is undertaken to summarize available data and compile all the updated information on its phytochemical and pharmacological activities. The herb has been known since ancient times for its curative properties and has been utilized for treatment of various ailments, treatment such as cancer, malaria, asthma, asthma, diarrhea, and skin problems. A wide range of chemical compounds including alkaloids, flavonoids, terpenoids, and tannins have been isolated from the plant. Extracts from this plant have been found to possess various pharmacological activities. This contribution provides a comprehensive review of its ethnomedical uses, chemical constituents and the pharmacological profile as a medicinal plant. Particular attention has been given to analgesic, anti-inflammatory, anti-cancer, anti-diabetic, anti-hyperlipidemic, anti- asthmatic, anxiolytic, antibacterial and other effects presented in this review such that the potential use of this plant in pharmaceutics can be evaluated. KEYWORDS: Traditional medicine, scholaris, Araceae, Pharmacological activities, Ethno medical uses.

INTRODUCTION Herbal medicines are being used by nearly about 80% of the world population, primarily in developing countries for primary health care [1]. Assessing the current status of health care system in adequacies of synthetic drugs are likely to be more glaring in the coming years. It has been reported that there has been an alarming increase in number of diseases and disorders caused by synthetic drugs prompting a switch over to traditional herbal medicine [2]. India has over 1,08,276 species of bacteria, fungi, animals and already identified and described [3]. Out of these about 84% species constitutes fungi (21.2%), flowering plants (13.9%) and insects (49.3%) [4]. It is a well-known fact that Traditional Systems of medicines always played important role in meeting the global health care needs. They are continuing to do so at present and shall play major role in future also. The system of medicines which are considered to be Indian in

Rakesh et al. / Pharma Science Monitor 11(1), Jan-Mar 2020, 19-39 Impact factor: 0.3397/ICV: 4.10 ISSN: 0976-7908 20 origin or the systems of medicine, which have come to India from outside and got assimilated in to Indian culture are known as Indian Systems of Medicine. India has the unique distinction of having six recognized systems of medicine in this category. They are Ayurveda, Siddha, Unani and Yoga, naturopathy and homoeopathy [5]. Among them, Ayurveda is being practiced for thousands of years. Considerable research on pharmacognosy, chemistry, pharmacology, and clinical therapeutics has been carried out on Ayurvedic medicinal plants. Natural products, including plants, animals, and minerals have been the basis of treatment of human diseases. The current accepted modern medicine or allopathy has gradually developed over the years by scientific and observational efforts of scientists. However, the basis of its development remains rooted in traditional medicine and therapies [6]. Plants have played a significant role in maintaining human health and improving quality of human life for thousands of years and have served humans well as valuable components of medicines, seasoning, beverages, cosmetic and dyes. Herbal medicine is based on the premise that plants contain natural substances that can promote health and alleviate illness in recent times, focus on plant research has increased all over the world and large body of evidence has collected to show immense potential of medicinal plants used in various traditional system. There are many herbs which are predominantly used to treat cardiovascular problems, liver diseases, CNS disorders, digestive and metabolic disorders. Given their potential to produce significant therapeutic effect they can be useful as drug or supplement in the treatment/management of various diseases. Herbal drugs or medicinal plants, their extract and their isolated compounds have demonstrated spectrum of biological activities. Ethno pharmacological studies on such herbs or medicinally imported plants continue to interest investigators throughout the world [7]. Selection of scientific and systematic approach for the biological evaluation of plant products based on their use in the traditional systems of medicine forms the basis for an ideal approach in the development of new drugs from plants. One such plant is Alstonia scholaris, commonly known as Indian devil tree (Eng.) and kutaj kul (Sans.). It is a tall and perennial herbaceous plant growing throughout India. PLANT PROFILE Alstonia scholaris (Linn) R.B.r. (Fig. 1) belongs to family Apocynaceae. The Apocynaceae family is a family of flowering plants, which includes trees, shrubs, herbs, and lianas. The family, as currently recognized, pharmacological activities includes some 1500 species divided

Rakesh et al. / Pharma Science Monitor 11(1), Jan-Mar 2020, 19-39 Impact factor: 0.3397/ICV: 4.10 ISSN: 0976-7908 21 in about 424 genera. Many species are tall trees found in tropical rainforests, and most are from the tropics and subtropics, but some grow in tropical dry, xeric environments [7, 8]. Synonyms [9] Latin: Echites scholaris L. Mant. and Pala scholaris L. Robert Eng.: Blackboard tree, Indian devil tree, white cheesewood Hindi: Chattiyan Saittan kajat Gujarati: Saptaparni Malayalam: Yaksippala Tamil: Elilappalai Geographical Source It is an evergreen, tropical tree native found in to the Indian subcontinent and Southeast Asia. Fig.1. Whole plant of Alstonia scholaris It is also found in Nepal, Sri Lanka, Southeast Asia, Cambodia, Myanmar, Thailand, Vietnam, Indonesia, Malaysia, Papua New Guinea and Philippines [9]. Taxonomical classification [10] Kingdom: Plantae Division: Magnoliophyta Class: Magnoliopsida Order: Family: Apocynaceae Genus: Alstonia Species: scholaris

Properties and actions of the plant mentioned in Ayurveda [11] Gunna (properties): laghu (small) and snigdha (slimy) Rasa (taste) : tickt (bitter) and kashya (astringent) Virya (potency) : ushna (hot) Vipaka : katu Karma : vishamjvaraghna

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Botanical description Parts used: Bark, leaves and milky exudates [12]. Morphological description [13] Alstonia scholaris is a large evergreen tree up to 30 m in height with a straight often fluted and buttressed bole, about 110 cm in diameter. Leaves: The upper sides of the leaves are glossy, while the underside is grayish. Leaves occur in whorls of 3-10; the leathery leaves are narrowly obovate to very narrowly spathulate, lateral veins occur in 25-50 pairs. Base is tapering. Flower: Small greenish white numerous in umbellate panicles. Calyx 2.5-3 mm long pubescent, corolla tube is almost 8 mm long villous inside the mouth with a ring of hairs. Fruits: Very strongly scanted fruits follicles of 30-60 cm long. Seeds: 6 mm long, linear-oblong, flattened, rounded and with a fringe of hairs at both ends, the hairs longer than the seeds. Bark: Grayish brown, branchlets are copiously lenticellate rough, lenticellate abounding in bitter. It is also called as ‘dita bark’. Traditional uses The plant is used as a tonic, antiperiodic, anthelmintic, stimulant, carminative, stomachic and expectorant. It is a valuable remedy in chronic diarrhea and advanced stage of dysentery. The leaves roasted and pulverized and made into poutices, act as a use full local stimulant to unhealthy ulcers with four discharges. The juice of leaves with that of fresh ginger root or zedoary is administered to women after confinement. In the Konkan the bark is given in leprosy, an extract being prepared from the fresh bark and given in milk. The bark of AS in combination with other drug is prescribed for the snake bite (Sushruta, Yogaratnakara) and scorpion sting (Sushruta). The bark of the plant is traditionally used by many ethnic groups of North East India and other parts of the world as a source cure against bacterial infection, malarial fever, toothache, rheumatism, snakebite, dysentery, bowl disorder, etc. Also, the latex is used in treating coughs, sores and fever [13, 14]. . PHYTOCHEMISTRY Alstonia scholaris contains alkaloids, flavonoids, triterpenoids, and tannins as a major class of phytocomponents [9] PHYTOCHEMICAL INVESTIGATIONS Mistry D et al, (2016) performed phytochemical investigations of entire plant of Alstonia scholaris and compared qualitatively and quantitatively phytochemical constituents present in

Rakesh et al. / Pharma Science Monitor 11(1), Jan-Mar 2020, 19-39 Impact factor: 0.3397/ICV: 4.10 ISSN: 0976-7908 23 bark, stem and leaves of the plant. Qualitative phytochemical screening revealed the presence of alkaloids, saponins, terpenoids, flavonoids, phenolic compounds, tannins, steroids, and glycosides in bark, stem and leaf extracts. The glycosides, alkaloids, gums and mucilage were found in higher quantity in bark of A. scholaris as compared to that in stem and leaf. Further, in vitro antioxidant potential of extracts from bark of the plant was also analyzed. Both aqueous and/or methanolic extracts from bark of the plant showed potent total antioxidant activity. At every concentration studied, percentage of superoxide radicals scavenged by aqueous extracts from bark of the plant was higher even than those of standard gallic acid at respective concentrations. Similarly the results of DPPH free radical scavenging assay showed that aqueous extracts from bark of the plane better scavenged free radicals than the methanolic extract from bark of the plant as well as standard Ascorbic acid tested at respective concentrations. Alkaloids Alstonia scholaris contains a large groups of alkaloids, echitamine (ditamine), ditaine, echitenine (Fig. 2a) and echitamidine (Fig. 2b) together with triterpenes, α-amyrin (Fig. 2c) and lupeol (Fig. 2d). Alstonamine, a new indole alkaloid, isolated from leaves together with rhazimanine. Moreover in 1955, reserpine was isolated from the plant root and bark [16, 17].

Fig. 2a. Echitamine Fig. 2b. Echitamidine

Figure 2c. α-amyrin Figure 2d. Lupeol

Fig. 2. Structures of some alkaloids isolated from Alstonia scholaris Salim et al, (2004) performed isolation of a new indole alkaloid, akuammiginone (Fig. 3), and a new glycosidic indole alkaloid, echitamidine-N-oxide 19-O-beta-d-glucopyranoside,

Rakesh et al. / Pharma Science Monitor 11(1), Jan-Mar 2020, 19-39 Impact factor: 0.3397/ICV: 4.10 ISSN: 0976-7908 24 together with the five known alkaloids, echitaminic acid, echitamidine N-oxide, N(b)- demethylalstogustine N-oxide, akuammicine N-oxide, and N(b)-demethylalstogustine, from the trunk bark of AS, collected in Timor, Indonesia. The structures of all compounds were elucidated by spectroscopic methods [18].

Fig. 3. Akuammiginone Xiang-Hai Cai et al, (2007) isolated a pair of geometrically isomeric monoterpenoid indole alkaloids with a skeleton rearrangement and two additional carbons, named (19, 20) E- alstoscholarine (Fig. 4a) and (19, 20) Z-alstoscholarine (Fig. 4b) from the leaf extract of plant. Their structures were elucidated on the basis of spectroscopic methods and then confirmed by X- ray crystal diffraction [19].

Fig. 4a. (19, 20) E-alstoscholarine Fig. 4b. (19, 20) Z-alstoscholarine

Fig. 4 Structure of isomeric monoterpenoid Indole alkaloids isolated from Alstonia scholaris Moreover in 2008, he isolated some more alkaloids from Alstonia leaves by using silica gel column chromatography and used spectroscopic techniques (NMR, IR, UV and MS) for structural elucidation. In study total four picrinine-type monoterpenoid indole alkaloids, picrinine (Fig. 5a), picralinal (Fig. 5b), 5-methoxyaspidophylline and 5-methoxystrictamine were obtained

Rakesh et al. / Pharma Science Monitor 11(1), Jan-Mar 2020, 19-39 Impact factor: 0.3397/ICV: 4.10 ISSN: 0976-7908 25 from the leaves. This was the first report regarding presence of indole alkaloids in the plant [20].

Figure 5a. Picrinine Figure 5b. Picralinal

Fig. 5 Structure of Picrinine-type monoterpenoid alkaloids isolated from Alstonia scholaris

Tao Feng et al, (2009) showed six new monoterpenoid indole alkaloids, scholarisines B- G (Fig. 6), together with 15 known analogues, were isolated from the bark of the plant. Their structures were determined by 1D and 2D NMR spectra and MS analyses [20].

Fig. 6. Scholarisine Flavonoids Hui et al, (2009) isolated and investigated the flavonoids in leaves of AS for the first time. In study the chemical constituents were isolated by column chromatography and their structures were elucidated on the basis of spectroscopic evidences and physicochemical properties. Total eight flavonoids were isolated and identified as kaempferol (Fig.7a), quercetin (Fig.7b), isorhamnetin,kaempferol-3-0-beta-D-galactopyranoside,quercetin-3-0-beta-Dgalacto pyranoside,isorhamnetin-3-0-beta-D galactopyranoside, kaempferol-3-0-beta-D xylopyranosyl- (2-1)-0-beta-D-galactopyranoside,quercetin-3-0-beta-D-xylopyranosyl-(2-1)-0-beta-D galactopyranoside [22].

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Fig. 7a. Kaempferol Fig. 7b. Quercetin

Fig. 7. Structure of some flavonoids isolated from Alstonia scholaris Zuraida et al, (2017) isolated and identified the flavonoid compounds from Alstonia scholaris. In the study the crude aqueous extract of the plant bark was prepared and fractionated by liquid-liquid fractionation using n-hexane, chloroform and 70% ethanol. Ethanol and chloroform fraction were analyzed for antioxidant activity through radical scavenge DPPH method, while identification of flavonoids was investigated by HPLC method. The results showed that yield of ethanol fraction was higher (2.52%) than that of chloroform fraction (0.83%) respectively. Ethanol fraction had stronger antioxidant activity with IC50 value 73.53 µg/mL than that of chloroform fraction (445.96 µg/mL) respectively. Identification of flavonoids through HPLC showed that ethanol fraction contain rutin (5.0112 mg/gm), and quercetin (0.0470 mg/gm) higher than that in chloroform fraction with rutin content (0.9358 mg/g) and quercetin (0.0376 mg/g). Rutin is dominant in both fractions, and rutin in ethanol fraction was higher than that in ethanol fraction. Quercetin, and rutin are flavonoid compounds act as antioxidants [23]. Triterpenoids Wang et al, (2009) isolated 2, 3-Secofernane triterpenoids, alstonic acids A and B (Fig. 8a & 8b), were isolated from the plant leaves. Their structures were established from MS and NMR spectroscopic analysis and confirmed by single crystal X-ray diffraction analysis [24].

Fig. 8a Alstonic acid A Fig. 8b Alstonic acid B

Fig. 8. Structure of terpenoids isolated from Alstonia scholaris

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ETHNOPHARMACOLOGY Medicinal uses The tender leaves in form of poultice are good for ulcers and vitiater in the condition of vata and falgia. The plant is bitter astringent, acrid, thermogenic, digestive, laxative, febrifuge, antipyretic, galactogouge, stomachic, cardioprotective. It is used in fever, malarial fever, abdominal disorder diarrhea, dysentery, dyspepsia, chronic and foul ulcers, ashthma, bronchitis, cardiopathy, helminthiasis. Also it is used as appetizer, laxative, anthelmintic, good in disease of the heart, asthma, leucoderma, ulcers, and disease of the blood, “tridosha”, pains, and tumors [13]. PHARMACOLOGICAL REPORTS Analgesic and anti-inflammatory action Shang et al, (2010) evaluated the anti-inflammatory and analgesic activities of the ethanolic extract of the Alstonia scholaris leaves. In the study the analgesic activities were investigated using acetic acid-induced writhing, hot-plate and formalin tests in mice, while the anti- inflammatory activities were carried out in vivo and in vitro, including xylene-induced ear edema and carrageenan-induced air pouch formation in mice, and COX-1, COX-2 and 5-LOX inhibition. In in vitro tests, alkaloids exhibited inhibition of inflammatory mediators (COX-1, COX-2 and 5-LOX), which is accordant with results on animal models [25]. Further the screening was carried out with ethyl acetate and alkaloid fractions of the ethanol extract of plant. During that it was observed that the both the fractions reduced acetic acid-induced writhing response in mice, significantly. The ethanol extract, ethyl acetate and alkaloid fractions remarkably inhibited xylene-induced ear edema. Further the investigation was focused on the alkaloids fraction and three main alkaloids isolated from the alkaloids fraction, in different animal models. Alkaloids reduced acetic acid-induced writhing response, and xylene- induced ear edema in mice. In the hot-plate test, alkaloids did not increase the latency period of mice obviously. In the formalin test, alkaloids did not inhibit the licking time in first phase, but significantly inhibited the licking time in second phase of mice. Alkaloids increased significantly SOD activity and decreased levels of NO, PGE2 and MDA significantly, in air pouch mice model. Moreover, some alkaloids isolated from the plant leaf exhibited inhibition of COX-1, COX-2 and 5-LOX in vitro anti-inflammatory assay, which supported alkaloids as the bioactive fraction [25]. Anti-cancer action Keawpradub et al, (1997) evaluated the anti-cancer potential of methanolic extract of root bark of Alstonia scholaris, A. glaucescens, and A. macrophylla, collected from Thailand for their

Rakesh et al. / Pharma Science Monitor 11(1), Jan-Mar 2020, 19-39 Impact factor: 0.3397/ICV: 4.10 ISSN: 0976-7908 28 cytotoxic activity against two human lung cancer cell lines, MOR-P (adenocarcinoma) and COR- L23 (large cell carcinoma), using the SRB assay. At the end of study, the significant cytotoxic activity was exhibited by the extract of A. scholaris on both cell lines. Activity-directed fractionation led to the isolation of a novel indole alkaloid, O-methylmacralstonine, from the most active fraction of A. scholaris along with four known alkaloids, talcarpine, villalstonine, pleiocarpamine, and macralstonine. The bisindole, villalstonine was found to possess 3 pronounced activity on both cell lines with an IC50 value less than 5 µM, but was about 10 times less potent than vinblastine sulphate. The monomeric alkaloid, talcarpine, was found to be inactive. Pleiocarpamine, O-methylmacralstonine and macralstonine were all considerably less active than villalstonine. Thus it was the first report, which showed that Alstonia scholaris posseses anti-cancer action [26]. Jagetia et al, (2004) investigated the anti-cancer chemomodulatory activity of the plant extract. The study was performed in combination with berberine hydrochloride (BCL), a topoisomerase inhibitor, in Ehrlich ascites carcinoma-bearing mice. The tumor-bearing animals were injected with various doses of extract, and 8 mg/kg of BCL (one-fifth of the 50% lethal dose) was combined with different doses of the methanol extract (60–240 mg/kg). The combination of 180 mg/kg of extract with 8 mg/kg of BCL showed the greatest antitumor effect; the number of tumor-free survivors was more, and the median survival time and the average survival time increased up to 47 and 40.5 days, respectively, when compared with either treatment alone. Similarly, when 180 mg/kg of extract was combined with different doses of BCL (2–12 mg/kg), a dose-dependent increase in the anti-cancer activity was observed up to 8 mg/kg of BCL. However, a further increase in the BCL dose to 10 and 12 mg/kg resulted in toxic side effects. The best effect was observed when 180 mg/kg of extract was combined with 6 or 8 mg/kg of BCL, where an increase in the antineoplastic activity was reported. The efficacy of the combination of 180 mg/kg of extract was also tested with 6 mg/kg body weight of BCL in various stages of tumor genesis, and it was effective when given in the early stages, although the efficiency decreased with an increase in the tumor developmental stages [27]. Further in 2006 he evaluated in vitro anticancer effect of various doses of an alkaloid fraction of the plant, in cultured human neoplastic cell lines (HeLa, HepG2, HL60, KB and MCF- 7) and in Ehrlich ascites carcinoma bearing mice. It was found that the treatment of HeLa cells with 25 µg/ml fraction resulted in a time dependent increase in the antineoplastic activity and the greatest activity was observed when the cells were exposed to alkaloidal fraction for 24 h. However, the exposure of the cells to alkaloid fraction for 4 h resulted in 25% viable cells and

Rakesh et al. / Pharma Science Monitor 11(1), Jan-Mar 2020, 19-39 Impact factor: 0.3397/ICV: 4.10 ISSN: 0976-7908 29 hence this time interval was considered to be the optimum time for treatment and further studies were carried out using this time. Treatment of various cells with alkaloidal fraction resulted in a concentration dependent decline in the viable cells. The IC50 was found to be 5.53, 25, 11.16, 10 and 29.76µg/ml for HeLa, HePG2, HL60, KB and MCF-7 cells, respectively. Similarly, administration of alkaloid fraction once daily for 9 consecutive days to the tumor bearing mice caused a dose dependent remission of the tumor up to 240 mg/kg body weight, where the greatest antitumor effect was observed. Since 240 mg/kg alkaloidal fraction showed toxic manifestations, the next lower dose of 210 mg/kg was considered as the best effective dose, in which 20% of the animals survived up to 120 days post-tumor-cell inoculation as against no survivors in the saline treated control group. The treatment resulted in a dose dependent elevation in the median survival time (MST) and the average survival time (AST) up to 240 mg/kg fraction and declined thereafter. The surviving animals were healthy and disease free. The effect of alkaloid fraction was better than cyclophosphamide, which was used as a positive control, where all the animals succumbed to death by 40 days and the MST and AST were 19.5 and 18.3 days, respectively. The effective dose of 210 mg of ASERS was 3/10 of the LD50 dose, which increased the MST and AST up to 54 and 49.5 days, respectively [28]. Chaudhary et al, (2009) investigated the possible chemopreventive and anti-oxidative properties of the plant on two-stage process of skin carcinogenesis induced by a single application of 7, 12-dimethyabenz (a) anthrecene (100 g/100 lit. acetone), and two weeks later, promoted by repeated application of croton oil (1% in acetone/thrice a week) till the end of the experiment (16 weeks) in Swiss albino mice. The tumor incidence, tumor yield, tumor burden and cumulative number of papillomas were found to be higher in the carcinogen treated control (without treatment) as compared to experimental animals (extract treated). Furthermore, a significant increase in reduced glutathione, superoxide dismutase and catalase but decrease in lipid peroxidation was measured in the extract administered experimental groups than the carcinogen treated control. The present study demonstrates the chemopreventive potential of plant bark extract in DMBA-induced skin tumorigenesis in Swiss albino mice [29]. Bagheri G et al, (2016) investigated Cytotoxic and Antioxidant Activities of Alstonia scholaris, Alstonia venenata and Moringa oleifera. In the study they prepared hexane, benzene, isopropanol, methanol, and water extracts of the selected plants. Cytotoxicity assays were determined by Trypan blue exclusion, MTT, and apoptotic methods. Antioxidant activities were assayed by superoxide scavenging, hydroxyl radical scavenging, and lipid peroxidation. The results showed that among the extracts tested for cytotoxicity on DLA cells, the most active

Rakesh et al. / Pharma Science Monitor 11(1), Jan-Mar 2020, 19-39 Impact factor: 0.3397/ICV: 4.10 ISSN: 0976-7908 30 extracts from A. scholaris and A. venenata were selected (extracts from M. oleifera did not show 100% cytotoxicity even at a dose of 500 µg/mL, so it was not considered for determining

EC50 value), and their EC50 values were determined. Among the extracts of A. scholaris, hexane extract of stem bark showed an EC50 value of 68.75 µg/mL, while n-hexane extract of the leaves showed a higher EC5 value of 118.75 µg/mL. A. venenata showed significant in vitro superoxide scavenging activity, superior to that of quercetin. A. venenata showed less superoxide scavenging activity. The IC50 values of hexane extract (A. scholaris), isopropanol extract (A. venenata), and quercetin were 90.5 ± 6.2, 7.5 ± 1.2, and 31.5 ± 2.5 µg/mL, respectively. So the study indicates that the plant can be a source of new generation of anticancer drugs to combat diseases such as lymphoma and leukemia. These can also be used as antioxidants in dietary supplements [30]. Further Ahmad MS et al, (2016) performed in vivo and in vitro methods using chromosomal aberrations (CAs), sister chromatid exchange (SCE) and replication index (RI) as markers, exposed by methyl methanesulfonate (MMS) as well as alcoholic extract of Alstonia scholaris in five increasing concentrations (200, 250, 300, 350 and 400 mg/kg body weight for in vivo and 150, 200, 250 and 300 μg/ml of culture) and of three different durations of 24, 48 and 72 h in the presence as well absence of S9mix. In the study the extracts of Alstonia reduces the total aberrant cells ranges from 10.0% to 41.84% and frequencies of aberration in the aberrant cells ranges from 220 to 124 against 290 aberrations causes due to MMS in vivo. Similarly in the in vitro, it reduces CAs (39.62%, 32.83%, and 38.48%) and (45.31%, 44.46%, and 38.34%) at 24, 48, and 72 h of exposure respectively; in the absence as well as presence of liver S9 fraction. It also reduces SCE from 7.70 to 4.20 per cell and enhances RI from 1.45 to 1.64. Thus the study concludes that the plant significantly reduces the number of aberrant cells and frequency of aberration per cell at each concentration and duration of exposure in vivo; and CAs and SCE in vitro and enhances RI [31]. Antimalarial action Gandhi et al, (1990) evaluated the antimalarial activity of petroleum ether extract and methanol extract of the bark of the plant. The study was performed on mice, infected with Plasmodium berghei. At the end of study, a dose-dependent improvement of conditions and delayed mortality was noticed amongst mice receiving methanol extract of plant as compared to pet. ether extract [32]. Moreover Keawpradub et al, (1999) observed that methanol extracts prepared from various parts of Alstonia scholaris collected from Thailand, possesses antiplasmodial activity

Rakesh et al. / Pharma Science Monitor 11(1), Jan-Mar 2020, 19-39 Impact factor: 0.3397/ICV: 4.10 ISSN: 0976-7908 31 against multidrug-resistant K1 strain of Plasmodium falciparum cultured in human erythrocytes. The results of the study showed pronounced antiplasmodial activity with methanol extract of the root bark of plant with an IC50 value of 5.7 micrograms/ml. Moreover total thirteen indole alkaloids were isolated from the active extract. Those alkaloids and a semisynthetic bisindole-O- acetylmacralstonine were subsequently tested against the K1 strain of P. falciparum. Pronounced antiplasmodial activity was observed mainly among the bisindole alkaloids, particularly villalstonine and macrocarpamine with IC50 values of 0.27 and 0.36 µm, respectively. The potent alkaloids were further tested against T9-96, the chloroquine-sensitive strain of P. falciparum. The results showed that the active alkaloids, in contrast to chloroquine, have significantly higher [33] affinity to the K1 strain than to the T9-96 strain . Anti-asthmatic activity (Bronchodilatory action) Channa et al, (2005) performed screening of the ethanol extract of the plant leaves to evaluate bronchodilatory activity in anaesthetized rats with the probable involvement of prostaglandins. However, in vitro preparations of guinea-pig trachea did not confirm this property, indicating that bronchodilation is not due to the direct tracheal smooth muscle relaxation. The vasodilatory activity of the extract was independent of adrenergic or muscarinic receptors or prostaglandins but was mainly via endothelial-derived relaxing factor, nitric oxide. The extract inhibited the spontaneous movements of rabbit jejunum and contractile effects of acetylcholine and histamine on guinea-pig ileum. Additionally, the extract caused marked reduction of barium chloride-, potassium chloride- and calcium chloride-induced contraction on guinea-pig ileum and pulmonary artery, implying a direct interference of plant extract with the influx of calcium ions into cells. However, the extract has no detectable effect on mobilization of intracellular calcium. These results coupled with the in vivo effects of ethanol extract revealed that the Alstonia scholaris leaves possess broncho-vasodilatory activity mediated presumably by prostaglandins, calcium antagonism and endothelium-derived relaxing factor [34]. Shang et al, (2010) evaluated the anti-tussive and anti-asthmatic activities of the ethanolic extract, fractions and main alkaloids of the plant leaves. The anti-tussive activity was evaluated using three different models including ammonia or sulfur dioxide induced mice coughing, and citric acid induced guinea pigs coughing. The anti-asthmatic activity was investigated on guinea pigs bronchoconstraction induced by histamine. The expectorant activity was evaluated by volume of phenol red in mice's tracheas. The alkaloids fraction significantly inhibited mice's frequency of cough induced by ammonia, increased mice's latent period of cough induced by sulfur dioxide, and increased guinea pigs' latent period of cough and inhibited

Rakesh et al. / Pharma Science Monitor 11(1), Jan-Mar 2020, 19-39 Impact factor: 0.3397/ICV: 4.10 ISSN: 0976-7908 32 frequency of cough. Besides, the alkaloids fraction increased delitescence of convulsion, and tumbles of guinea pigs in anti-asthmatic test, and enhanced tracheal phenol red output in expectorant evaluation. Moreover, the main alkaloid, picrinine exhibited anti-tussive and anti- asthmatic activities in vivo. Thus the alkaloid fraction of the plant extract was observed to have anti-tussive, anti-asthmatic and expectorant activities [35]. Immunostimulating property Soemardji et al, (2000) evaluated the immunostimulating effect of the plant bark extracts in BALB/c mouse. In the study the extracts were administered orally, once a day for 7 consecutive days. The results showed that at the same doses (50, 100 and 200 mg/kg body wt.) the aqueous extract had higher phagocytic index (1.39-1.79) than the ethanolic extracts (0.81-0.93) in normal mice. The aqueous extract at 50 mg/kg also enhanced phagocytic activity of immunosuppressed mice significantly (p<0.01). At 50 and 100 mg/kg the extract prevents the decrease of immune system induced by prednisone. The aqueous extract at 100 mg/kg increased lytic activity of peritoneal exudate cells against Escherichia coli significantly (p<0.05). At the doses of 50 and 100 mg/kg the aqueous extract had no effect on primary antibody level. The aqueous extract at 50 mg/kg induced the cellular immune response while at 100 mg/kg inhibited the delayed type of hypersensitivity reaction [36]. Anti-diarrhoeal and spasmolytic action Shah et al. (2010) evaluated the antidiarrhoeal and antispasmodic activities of the plant by using in vivo and in vitro techniques. In the in vivo study the crude extract of plant provided 31-84% protection against castor oil-induced diarrhoea in mice at 100-1000 mg/kg doses, similar to loperamide. In isolated rabbit jejunum preparation, the plant extract inhibited spontaneous and + high K (80 mm)-induced contractions, with respective EC50 values of 1.04 (0.73-1.48) and 1.02 mg/ml (0.56-1.84; 95% CI), thus showing spasmolytic activity mediated possibly through calcium channel blockade (CCB). The CCB activity was further confirmed when pretreatment of the tissue with the extract (0.3-1 mg/ml) caused a rightward shift in the Ca++ concentration-response curves (CRC) similar to a standard calcium channel blocker. Loperamide also inhibited spontaneous and high K+ precontractions as well as shifted the Ca++ CRCs to the right. These results indicated that the crude extract of the plant possesses antidiarrhoeal and spasmolytic effects, mediated possibly through the presence of CCB-like constituents and this study provides a mechanistic base for its medicinal use in diarrhoea and colic [37]. Neuroprotective potential Anti-stress and Nootropic (cognition) action

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Kulkarni et al, (2009) evaluated the stress condition and its modulation by the methanolic extract of AS bark of, using acute restraint stress model in mice. The extract was also evaluated for nootropic and antioxidant potential to support anti-stress activity testing. Acute restraint stress resulted in significant increase of plasma corticosterone, glucose, protein, cholesterol and triglyceride levels in stress group of animals. Methanolic extract pretreatment at 100, 250 and 500 mg/kg for 7 days displayed promising anti-stress effect by normalizing these stress-induced biochemical perturbations in plasma of mice. Effect on cognitive functions was evaluated using passive avoidance model and elevated plus maze model. Pretreatment with extract at 100, 250 and 500 mg/kg augmented acquisition and retention of memory of learned task as evidenced by increased step-down and shortened-transfer latency in passive avoidance model and elevated plus maze model, respectively. Diazepam (2 mg/kg, ip) and piracetam (200 mg/kg, po) were used as standard drugs for anti-stress and nootropic activity testing. Further, the extract at 200 µg/ml showed maximum scavenging of stable radical 1,1-diphenyl, 2-picryl hydrazyl at 90.11% and nitric oxide radical at 62.77%. The present study provided the scientific support for anti- stress (adaptogenic), antioxidant and nootropic activities of the methanolic extract of bark of the plant [38]. Anxiolytic activity Sinnathambi A et al, (2013) evaluated anti-anxiety activity of the ethanolic extract of leaves of Alstonia scholaris. In the study, they fractionated the ethanolic extract of leaves of A. scholaris leaves with Dichloromethane fraction (DCM). DCM fraction (10 and 30 mg/kg) was tested against various anti-anxiety models viz. elevated plus maze, open field, hole board, light dark test, mirror chamber and foot shock induced aggression models. The false positive results were overruled by actophotometer and rotarod tests. The change in brain monoamines was estimated. The possible dopaminergic effect was tested by haloperidol-induced catalepsy and 6-hydroxy dopamine induced Parkinsonism. The DCM fraction was found to be significantly active in all the tested models of anxiety, which confirmed their anti-anxiety activity. Lack of significant change in locomotion test indicated that DCM did not have stimulant or sedative effects. There was also no significant change in the time spent in the rota rod, which indicated its lack of motor or muscle in coordination. Increase in brain dopamine levels and reversal of dopamine-induced catalepsy confirmed that the mechanism of anti-anxiety activity is mediated through dopamine. The effect of DCM in 6-hydroxy dopamine-induced anxiety was conclusive of its dopaminergic activity. Thus the present study indicates that the plant possesses anti-anxiety and the apparent mechanism of action is through dopaminergic mediation [39].

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Neuroleptic activity Jash R & Chowdary AC, (2014) evaluated the neuroleptic activity of Alstonia Scholaris along with Bacopa Monnieri with different anti-psychotic animal models with a view that these plant extracts shall have no or at least reduced adverse effect so that it can be used for long duration. In the study two doses of the ethanol extracts of both the plants (100 and 200 mg/kg) and also standard drug haloperidol (0.2 mg/kg) were administered to their respective groups once daily with 5 different animal models. After that, the concentration of the dopamine neurotransmitter was estimated in two different regions of the brain viz. frontal cortex and striatum. The result of the study indicated a significant reduction of amphetamine-induced stereotype and conditioned avoidance response for both the extracts compared with the control group, but both did not have any significant effect in phencyclidine-induced locomotor activity and social interaction activity. However, both the extracts showed minor signs of catalepsy compared to the control group. The study also revealed that the neuroleptic effect was due to the reduction of the dopamine concentration in the frontal cortex region of the rat brain. The results largely pointed out the fact that both the extract may be having the property to alleviate the positive symptoms of schizophrenia by reducing the dopamine levels of dopaminergic neurons of the brain [40]. Anti-fertility action Gupta et al. (2005) evaluated the anti-fertility activity of lupeol acetate, isolated from benzene extract of the plant in male albino rats. The results of the study revealed that the treatment with lupeol acetate at the dose level of 10 mg/rat/day did not cause any significant change in the body weights, but significant reduction in the weight of reproductive organs, i.e. testes, epididymides, seminal vesicle and ventral prostate, was observed. Testicular sperm count, epididymal sperm count and motility were found significantly declined when compared with controls, which resulted in reduction of male fertility by 100%. Arrest of spermatogenesis was noted at various stages with production of primary spermatocytes (preleptotene and pachytene), secondary spermatocytes and step-19 spermatids were decreased by 52.36, 54.91, 55.67 and 69.65%, respectively. The seminiferous tubules appeared reduced in size by 24.62%. Cross-sectional surface area of sertoli cells as well as their counts was found to be significantly depleted. Leydig cell nuclear area and number of mature Leydig cells were decreased by 27.65 and 35.47%. Biochemical parameters of tissues i.e. protein, sialic acid, glycogen and cholesterol content of testes and seminal vesicular fructose also showed significant reduction [41]. Antibacterial potential

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Khyade et al, (2009) examined antibacterial activity of the methnolic extract of the plant against Gram +ve as well as -ve organisms. The results exhibited broad-spectrum antibacterial activity of the plant against tested organisms. Maximum activity was exhibited against Bacillus subtilis followed by Escherichia coli and Staphylococcus aureus. Chloroform and acetone leaf extracts exhibited lesser activity, while petroleum ether extract showed no inhibition [42]. Anti-oxidant potential Jagetia et al, (2004) evaluated the anti-oxidant action of the plant along with other 17 well known Indian medicinal plants for their possible regulatory effect on nitric oxide (NO) levels using sodium nitroprusside, as an NO donor in vitro. In results it was found that most of the plant extracts demonstrated direct scavenging of NO and exhibited significant activity. Moreover it was observed that the plant bark showed the greatest NO scavenging effect of 81.86 % at 250 mg/ml, as compared to other plants [43]. In addition Jain et al., (2010) evaluated the in vitro free radical scavenging activity of the petroleum ether, ethanol and aqueous extracts of the plant. Each extract was examined to study the free radical scavenging activity by superoxide scavenging assay method. In results it was observed that all the extracts showed the dose- and time-dependent inhibition of the superoxide scavenging activity [44]. Antidiabetic and Antihyperlidemic activity Bandawans D et al, (2011) investigated antidiabetic and antihyperlidemic effect of aqueous extract of Alstonia scholaris bark in streptozotocin (STZ) induced diabetes in rats. During the study the diabetes was induced by single dose of STZ (65 mg/kg) in citrate buffer, while the normal control group was given the vehicle (citrate buffer) only. After three days of induction of diabetes, the diabetic animals were treated further four weeks with aqueous extract of plant bark (150 mg/kg and 300 mg/kg) and glibenclamide (4 mg/kg). Blood glucose estimation was performed every week of the study. At the end of study period, animals were sacrificed for biochemical studies. STZ-induced diabetic rats showed marked hyperglycemia, hypertriglyceridemia and hypercholesterolemia at the end of study period. Body weight and liver glycogen levels were reduced and glycosylated haemoglobin levels were significantly increased in diabetic rats. The four week treatment with aqueous extract of plant bark (150 mg/kg and 300 mg/kg) significantly ameliorated the alterations in fasting blood glucose, serum triglyceride, serum cholesterol, liver glycogen, glycosylated haemoglobin and body weight in diabetic rats. Thus the present study suggests the potential of plant bark exhibits antidiabetic and antihyperlipidemic properties [45].

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Moreover Wanjari AD et al, (2019) carried out a study to evaluate the influence of alcoholic and aqueous extracts of leaves of Alstonia scholaris R Br. on α amylase inhibition activity by in-vitro antidiabetic model. The study results that among the aqueous and alcoholic extract, the alcoholic extract of plant leaves exhibited significant α amylase inhibition in vitro. Thus the study reveals the significant α amylase inhibitory potential of the plant [46]. Some Ayuervedic Preparations of Alstonia scholaris Tincture, extract, powder and decoction [11] DISCUSSION Alstonia scholaris is a well-known plant used in the Indian traditional system of medicine, besides which folklore medicine also claims its uses especially in cancer, malaria, asthma, diarrhoea, and etc. Recently Alstonia scholaris fruit is widely cultivated throughout the world for its medicinal uses. It is also a very important plant in a number of diseases and for that we have enough scientific reports and data. Chemically the plant contains various biologically active phytoconstituents like alkaloids, flavonoids, triterpenes, and tannins. Thus Alstonia scholaris is considered as a very important gift of Ayurveda to mankind. REFERENCES 1. Kamboj VP: Herbal medicine. Current Science Journal 2000; 78(1): 35-39. 2. Ghule SP, Patil DK: Kisan World 2001: 28(10); 33-34. 3. Khosoo TN: India’s biodiversity: Tasks ahead. Current Science Journal 1994, 67(8); 577- 582. 4. Goldberg B: Alternative medicine: The definitive guide. Future Medicine Publishers, Puyallup, Washington 1994. pp. 257. 5. Prasad LV, Chaudhury RR, Rafei UM: Indian System of Medicine and Homoeopathy Traditional Medicine in Asia. In: editors. New Delhi: WHO- Regional Office for South East Asia 2002. pp. 283-286. 6. Vishnukanta and Rana AC: Melia azedarach: Pharmacognosy reviews 2008; 2(3):173- 179. 7. Arulmozhi S, Mazumber PM, Ashok P, LS Narayanan LS: Pharmacological activites of Alstonia scholaris Linn. (Apocynaceae) – A Review. Pharmacognosy Reviews 2007; 1(1): 163-170. 8. Endress PV and Bruyns. A revised classification of the Apocynaceae. Botanical Reviews 2000; 66: 1-56. 9. Khare CP: Encyclopedia of Indian Medicinal Plants, Springer Publications 2008. pp. 38

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35. Shang JH, Feng X, Zhao YL,Wang K, Zhang Y, Luo Y: Pharmacological evaluation of Alstonia scholaris: anti-tussive, anti-asthmatic and expectorant activities. Journal of Ethnopharmacology 2010; 129(3): 293-298. 36. Soemardji AA, Iwo MI, Retnoningrum DS: Immunostimulating effect of pule (Alstonia scholaris L. R.Br., Apocynaceae) bark extracts. Clinical Hemorheology and Microcirculation 2000; 23(2): 177-183. 37. Shah AJ, Gowani SA, Zuberi Aj, Ghayur MN, Gilani AH: Antidiarrhoeal and spasmolytic activities of the methanolic crude extract of Alstonia scholaris L. are mediated through calcium channel blockade. Phytotherapy Research 2010; 24(1): 28-32. 38. Kulkarni MP and Juveka AR: Effect of Alstonia scholaris (Linn.) R. Br. on stress and cognition in mice. Indian Journal of Experimental Biology 2009; 47(1): 47-52. 39. Sinnathambi A, Mazumder PM, Lohidasan S, Thakurdesai P: Dopaminergic effect of Alstonia scholaris Linn. R.Br. in experimentally induced anxiety. Biomedicine and Aging Pathology 2013, 3(2); 51-57. 40. Jash R, Chowdary AC: Ethanolic extracts of Alstonia Scholaris and Bacopa Monniera possess neuroleptic activity due to anti-dopaminergic effect. Pharmacognosy Research 2014; 6(1): 46-51. 41. Gupta RS, Bhatnager AK, Joshi YS, Sharma MC, Khushalani V: Induction of antifertility with lupeol acetate in male albino rats. Indian Journal of Pharmaceutical Sciences 2005; 75(2): 57-62. 42. Khyade MS and Vaikos NP: Phytochemical and antibacterial properties of leaves of Alstonia scholaris R.Br. African Journal of Biotechnology 2009; 8(22): 6434-6437. 43. Jagetia GC and Baliga MS: The Evaluation of Nitric Oxide Scavenging Activity of Certain Indian Medicinal Plants in Vitro: A Preliminary Study. Journal of Medicinal Food 2004; 7(3): 343-348. 44. Jain S, Jain A, Jain N, Jain DK, Balekar N: Phytochemical investigation and evaluation of in vitro free radical scavenging activity of Tabernaemontana divaricata Linn. Natural Product Research 2010; 24(3): 300-304. 45. Bandawans D, Juvekar A, Juvekar M: Antidiabetic and Antihyperlipidemic Effect of Alstonia Scholaris Linn Bark in Streptozotocin Induced Diabetic Rats. Indian Journal of Pharmaceutical Research and Education 2011; 45(2): 114-120. 46. Wanjari AD, Maske AO, Akare SC, Motghare RD, Sahare AY, Bodhankar SS and Nimbekar T P: Alpha amylase inhibition activity of Leaves of Alstonia scholaris R Br. International Journal of Pharmacological Research 2019; 09(04): e5194.

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