Ethnomedicinal Use and Phytochemical Analysis of Selected Medicinal Plants of Mizoram, India

Mizoram is hot spot of plant biodiversity and treasure for several medicinal plants. The elders of Mizoram use several plants for medicinal purposes and treatment of many human diseases. The present study was undertaken to document the ethnomedicinal uses of twenty plants and analyse their phytochemical constituents. The leaves of Ageratum conyzoides Linn. (Family: Compositae), Ananas cosmosus (L) Merr), Blumea balsamifera DC, Blumea lanceolaria Roxb., Cannabis sativa Linn., Carica papaya Linn., Clerodendrum colebookianum Walp., Dillenia pentagyna Roxb., Dysoxylum gobara Buch.-Ham, Eryngium foetidium Linn., Hedyotes scandens Roxb., Helicia excelsa Roxb., Mikania micranta Kunth. Mimosa pudica Linn, Polygonum chinanse Linn, Smilax ovalifolia Roxb., stem bark of Oroxylum indicum Vent., Schima wallichii (DC) Korth. and the whole plant of Callicarpa arborea Roxb., and Centella asiatica Linn., were dried and powdered. The alkaloid, flavonoid, cardiac glycoside, saponin, tannin, triterpenoid and phlobatannin contents in all these plants were estimated using standard methods. It was observed that all twenty plants contained alkaloids, flavonoids, saponins and tannins. The cardiac glycosides were present in all plants except B. lanceolaria, C. sativa, and C. bookenium. Ten plants showed the presence of phlobatannins and only four out of twenty plants showed the presence of all the phytochemicals screened. Our study indicates that the all ethnomedicinal plants selected in the present study showed the presence of phytochemicals like flavonoids, alkaloids, saponins, tannins, phlobatannins, terpenoids and cardiac glycosides and their medicinal properties may be due to the presence of these important phytochemicals.


Introduction
Plants have attracted the attention of humans since the advent of man on the earth. Plants also find innumerable uses in the human civilization since its inception. The plants also find their use as medicine in human healthcare. Several traditional systems have evolved in the world, which use plants to cater to the needs of healthcare and they are still in practice around the world. The use of plants and natural products received a fillip when World Health Organization recognized plant and natural products based medicinal systems as alternative and complimentary therapy in the year 2002 [1]. The use of medicinal plants for human healthcare is well documented in India, China, Egypt and Arab world [2]. The utility of plants and natural products was realized in India since Vedic times that resulted in the development of system of medicine the Ayurveda, that describes various uses of botanicals and other natural products in the treatment of several diseases and this knowledge has been compiled as several samhitas. The Chark samhita and other texts give a threadbare account of various ailments and their treatment strategies using mainly natural products and plants as medicines [3].
It is also well known that there are several chronic diseases which cannot still be treated by the modern system of medicine and humans are left with no choice except to rely on the age old system of traditional medicine. The traditional system of medicine prescribes drug as single plant products or a mixture of several plants depending on the disease, which are mainly administered orally. Many of the herbs, oils and potions are Trends in Green Chemistry also applied topically depending on the disease [4]. The use of plants as medicine has come a long way of centuries of experimentation on humans based on trial and error leading to development of many traditional systems of medicine that are in vogue in the modern world [5]. Systematic research has led to the development of several modern day drugs from plants. It is known that 25% of modern drugs are derived from the plants and in case of cancer treatment this figure is as high as 75% [6]. The role of natural products in drug discovery can be understood by the fact that nearly 100 natural products or natural product derived drugs had entered various phases of clinical trials in the year 2013 [7].
Traditional healers have used several plants to treat various diseases over the years and use of many of these plants as medicine is described in Ayurvedic texts [8]. Many diseases including cancer, leprosy, syphilis, all types of fever, children's abdominal disorders, elephantiasis and hydrocele have been treated using botanical sources [9]. The plants synthesize several non-nutritive phytochemicals for different purposes and these have been reported to be beneficial as they possess various medicinal activities in humans [5]. These phytochemicals are primary or secondary metabolites with varying biological activities and they are usually the main active principles present in plants [10]. The primary metabolites are essential for the sustenance of normal plant's life, whereas the secondary metabolites including tannins, saponins, flavonoids, alkaloids, terpenoids, glycosides synthesized by plants for other purposes and are non-nutritive [5,11]. Despite the fact that they are nonnutritive these secondary metabolites play a crucial role in the life cycle of plants. They provide attractive colours to flowers, help in pollination, protect against the stress, and ward off against the predators and insects [12].
Many of the secondary metabolites are important therapeutic agents in humans, treatment of animal diseases, crop production, scientific research and numerous other areas [13]. It is also well known that around 80% of the world's population mainly relies on plants and natural products for their healthcare requirements, which emphasizes the need to analyse the chemical constituents of plants and put their traditional use on firm scientific footing. Considering the importance of non-nutritive phytochemicals in human healthcare it was desired to study the phytochemical constituents of various ethnomedicinal plants traditionally used as medicine in Mizoram, India.

Chemicals and reagents
Potassium iodide, bismuth nitrate, sulphuric acid, ferric chloride, hydrochloric acid, aluminium chloride, ammonium hydroxide, glacial acetic acid, chloroform, and olive oil were procured from Sd fine Chemical Ltd., Mumbai, India.

Preparation of extracts
Five grams (5 g) each of the plant powder was weighed and transferred into a beaker containing 200 ml of distilled water. The mixture was heated on a hot plate with continuous stirring at 60°-80°C for 30 minutes. The water extract was filtered through filter paper and the filtrate was used for the phytochemical analysis. The aqueous extracts were kept in refrigerator at 0°C until use.

Phytochemical analysis
The aqueous extract of all selected plants listed above were subjected to different phytochemical tests using standard procedures to identify the phytochemical constituents qualitatively as described earlier [10,14-16].

Test for alkaloids
The presence of alkaloids in the aqueous extracts of different plants was determined by mixing 1 ml of each plant extract with Dragendorff's reagent. The formation of reddish brown precipitate indicated the presence of alkaloids.

Test for cardiac glycosides (Keller-Killani test)
Five ml of each extracts was treated with 2 ml of glacial acetic acid containing one drop of ferric chloride solution, and underlayed with 1 ml of concentrated sulphuric acid (H 2 SO 4 ). The appearance of a brown ring at the interface indicated a deoxysugar, which is characteristic of cardenolides.

Test for saponins
The presence of saponins in the aqueous extract of different plants was detected by mixing 2 ml of extract with 2 ml of distilled water and shaking it vigorously. The formation of a stable persistent froth indicated that the presence of saponins. The frothing was mixed with 3 drops of olive oil and shaken

Test for terpenoids (Salkowski test)
The terpenoids in aqueous extract of various plant were detected by mixing 5 ml of each extract with 2 ml of chloroform with the careful addition of 3 ml concentrated H 2 SO 4 so as to allow the formation of a layer. The formation of a reddish brown colour at the interface confirmed the presence of terpenoids.

Test for tannins
The presence of tannins in different plant extracts was ascertained by mixing 5 ml of each aqueous extract with a few drops of 0.1% ferric chloride. The formation of brownish green or a blue-black colour indicated the presence of tannins [21-24].

Test for phlobatannins
The phlobatannins were detected by mixing each plant extract with hydrochloric acid. The deposition of a red precipitate after boiling was considered as the evidence for the presence of phlobatanins.

Results
The results of folklore use and phytochemical analyses of various medicinal plants are presented in Tables 2 and 3.

Traditional uses
The elders of Mizoram were contacted and a discourse with them revealed different traditional uses of the plants listed above which are described below.

Ageratum conyzoides:
The fresh shoot juice of Ageratum conyzoides is applied on cuts and wounds for rapid healing.
The plant decoction mixed with pepper paste is administered in patients suffering from acute stomachache. The crushed leaves are also topically applied on itches, cuts and leprotic condition. The leaves and roots are used to cure cholera and stomach ulcers. The root juice and leaves are also used to treat skin diseases, caterpillar sting, opthalmia, fresh cuts and sores (Table 2).

Ananas cosmosus:
The fruit or fruit juice is administered in stomach ulcers, and typhoid to remove helminthic infection. A handful of white young leaves are given daily for at least one week to expel intestinal worms ( Table 2).
Blumea balsamifera: Decoction and infusion of the leaves and bark is used as an expectorant ( Table 2). Blumea lanceolaria: Infusion or decoction of the leaves is administered orally to treat stomach ulcers and the leaves are topically applied to cure wounds. The leaves are also given during indigestion, asthma, tuberculosis and chronic dysentery. The leaf juice is also topically applied in the condition of scabies, skin diseases, sores and dandruff ( Table 2).

Callicarpa arborea:
The bark juice of Callicarpa arborea is orally taken to cure stomach pain, dysentery and vomiting. Bark also finds its use for skin diseases, scorpion sting. The fever, gastric diseases, giddiness and headaches are treated by giving leaf juice or leaf infusion. It possesses tonic and carminative properties and is also used in cutaneous diseases. Decoction of the leaf and bark is used to treat internal bleeding, stomach pain, flesh wound, abrasion, diabetes, colic, stomach ulcers and diarrhoea (Table 2).

Cannabis sativa:
The whole plant is used to cure stomach pain, dysentery and as an analgesic. The dried leaves are also rolled like cigarettes and smoked (Table 2).
Carica papaya: Different parts of this plant are used for dysentery, jaundice, toothache, snake bite, ulcers, and eczema in the form of decoction or infusion (

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Dillenia pentagyna: The boiled leaves and roots are used to treat stomach ulcers and rheumatic pain. Decoction of the bark/leaves is given to treat gastric trouble, asthma, dysentery, and cancer ( Table 2).
Dysoxylum gobara: Decoction of leaves is used to cure diarrhoea and dysentery (Table 2).
Eryngium foetidium: Decoction of the whole plant is used in the treatment of ulcers, piles, and fever. The leaves are used to expel thread worms from the body and also as a remedy for food poisoning. The roots and leaves are boiled in water and are given for malarial fever, diabetes, pneumonia and constipation ( Table 2).
Hedyotes scandens: Decoction or infusion of the leaves or roots is used for sprain, skin diseases and fever. The whole plant decoction of Hedyotes scandens is used to treat swelling and kidney problems. Juice of the crushed leaves is also externally applied to cure sores, rheumatism and eye diseases ( Table 2).
Helicia excelsa: Decoction of the leaf or bark is taken orally in the conditions of fever, diarrhoea and stomachache ( Table 2).

Mikania micranta:
Pressed leaves are topically applied on fresh wounds and the infusion of the whole plant is given orally during stomach pain. Juice of the crushed leaves is used in fever, stomachache, diarrhoea, dysentery, insect bites, scorpion sting and also applied on fresh wounds. Leaf juice is used in dysentery and as a haemostatic agent. Leaves boiled with Vitex peduncularis are used to treat fever (Table 2).

Mimosa pudica:
Infusion or decoction of the whole plant is used as an antiseptic and for skin diseases. Decoction of the roots and leaves is given in liver and kidney diseases. Leaves and roots of Mimosa pudica are used in the conditions of piles and fistula. The root decoction is useful in gravelliest complaint (Table 2).
Oroxylum indicum: Decoction of the roots and bark is used for stomach ulcer, diarrhoea. The roasted pods are eaten in goitre. Decoction of the root bark is used to cure fever, colic, stomach ulcers, constipation, indigestion, intestinal worms, strangury, asthma, cough, hiccough, diarrhoea and dysentery. Poultice of the bark is topically applied during rheumatism, sprains, inflammations and skin diseases. Decoction of the leaves is also useful in headaches, flatulence, and ulcers. The fruits are used to treat colic, cough, heart diseases, bronchitis, dyspepsia, leucoderma and piles (Table 2).
Polygnum chinanse: Decoction or infusion of bark is used to treat arthritis, diarrhoea, asthma, piles, bronchitis orally ( Table 2). Schima wallichii: Decoction or infusion of leaf is used as an antiseptic. Fruit decoction of Schima wallichii is used to treat snake and insect bites. Bark powder is taken with water for the treatment of gastritis (Table 2).
Smilax ovalifolia: Decoction of the bark and root are used topically as anti-snake venom, and to treat cuts and wounds, and snake bite. It is given orally to treat stomach ulcers. The roots are given for dysentery and urinary complaints ( Table 2).
The cardiac glycosides were present in most of the plants except B. lanceolaria, C. sativa, and C. bookenium (Table 3). Of all the plants screened, only C. arborea, D. pentagyna, D. gobara and H.

Discussion
Use of botanicals for healthcare is as old as human civilization. They are considered non-toxic and safer than other exotic pure chemicals. This may be due to the fact that phytoceuticals origin is biological and also they have been experimented since the advent of human history [12,25]. The wide spread use of medicinal plants in healthcare entails that their systematic phytochemical evaluation shall be undertaken for the reasons of safety and medicinal use. Therefore, an attempt has been made to analyse phytochemical constituents of certain plants which have been used traditionally for human healthcare in Mizoram.  Cardiac glycosides are another class of phytochemicals synthesized by plants, which have several medicinal properties. The anticancer effects of cardiac glycosides were realized as early as 1967 and the recent preclinical investigation in HT29, HCT116, and CC2 colon cancer cell lines proved their cytotoxic action in vitro [43]. The cardiac glycosides have also been found to act against, melanoma, breast, lung, prostate, pancreatic cancers, leukaemia, neuroblastoma and renal adenocarcinoma [44]. The administration of a cardiac glycoside digoxin in combination with chemotherapy has been reported to increase the overall survival of patients receiving chemotherapy for the treatment of colorectal, breast, head and neck, and hepatocellular carcinoma [45]. The cardiac glycosides have been used in the treatment of cardiovascular diseases [46]. Most of the plants contained cardiac glycosides except B. lanceolaria, C. sativa, and C. colebookianum and many of the medicinal properties may be related to these phytochemicals.
All the plants screened have shown the presence of saponins that are a vast group of glycosides, differentiated by their surfaceactive properties from other group of glycosides. They possess the property of foam formation as well as detergent ability, which account for their soap like behaviour in water and hence they can be used as detergents [47][48][49]. They are allelopathic and act as defence against insects and pathogens in plants [50]. Saponins have been found to be antimicrobial, antimalarial, antiplasmodial, antiproliferative, antipsoriatic, antiallergic, antiatherosclerosis, antidiabetic, insecticidal, molluscicidal, anti-inflammatory and anticancer activities. The have also been reported to be active against obesity [51][52][53][54][55][56]. Some of the medicinal activities exhibited by the evaluated plants may be attributed to the presence of saponins in them.
Terpenoids, were detected in half of the plants screened, and they are classified into different types based on the number of union of their C5 isoprene units. They are the primary constituents of the essential oils in many plants and they are synthesized for defence or as signals against indirect defence including herbivory and other enemies [57]. Plant synthesize nearly 40,000 terpenoid molecules as secondary metabolites and they have diverse applications as industrial chemicals, flavouring agents, pharmaceuticals, fragrance, pesticides and disinfectants [58]. The terpenoids have been reported to show a diverse array of medicinal activities including, antiviral, antibacterial antimalarial, antiinflammatory, anticancer and chemopreventive. They have been found to inhibit cholesterol synthesis [59][60][61][62][63]. The medicinal properties shown by ten of the twenty plants may also be due to their terpenoid contents.
Tannins are complex polyphenolic phytochemicals, which are synthesized by numerous plants as secondary metabolites [64,65]. The tannins protect plants against the attack by herbivores and insects by decreasing the availability of proteins or inducing toxicity [66,67]. They act as a barrier for microorganisms and protect the plants due to their ability to form complexes with protein, starches and other macromolecules [68]. Tannins have been reported to act as antioxidants in vertebrates, and prooxidants in the presence of oxygen [67]. The tannins have been reported to act as astringent, antibacterial, antiulcerogenic, antiviral, antitumour, antithrombogenic, and antiinflammatory [69][70][71][72]. These observations confirm the medicinal uses of all plants selected in the present study, which may be due to the presence of tannins.
Phlobatannins are coloured phenolic compounds and could be detected only in eight out of twenty plants subjected to phytochemical analysis. They have been reported to possess wound healing, anti-inflammatory, antioxidant and analgesic activities [73][74][75]. This suggests that the leaf and bark of above tested plants might have useful applications in ethnomedicinal practices.