Pharmacognostical Standardization, Phytochemical Investigation, and Anthelminthic Activity of Arisaema propinquum Schott Rhizomes

Ethnopharmacology: Arisaema propinquum Schott ( Araceae ) is commonly known as cobra lily. Traditionally, the rhizomes were used as vermifuge, in rheumatism, as stomachache, and in snake bites. Objectives: The present study was designed to evaluate the pharmacognostical parameters and anthelminthic activity of A. propinquum rhizomes. Materials and Methods: The rhizomes of A. propinquum Schott was collected, shade dried and then powdered, and then evaluated for pharmacognostic parameters such as macro‑ and microscopical characters, physico‑chemical parameters, and phytochemical analysis using standard procedures. Anthelminthic activity of the extracts was elevated against Pheretima posthuma . Results: The rhizomes are light brown in color with pungent odor and astringent taste. Transverse section of rhizomes showed intercellular schizogenous cavities, xylem vessels, phloem vessels, and parenchymatous cells. Phytochemical screening of the extracts reveals the presence of alkaloids, carbohydrates, cardiac glycosides, coumarins, proteins, amino acids, phenols, tannins, flavonoids, saponins, steroids, and terpenoids. Physiochemical parameters including ash values showed 6.32% total ash, 1.77% acid insoluble ash, 5.15% water‑soluble ash, and 8.55% sulfated ash. Other parameters such as extractive value, foreign matter, moisture content, swelling index, foaming index, pH of different solvents, and fluorescence analysis were also determined. Both the methanolic and aqueous extracts of A. propinquum showed dose‑dependent anthelminthic activity against P. posthuma compared to standard albendazole. Conclusion: This is the first report on the pharmacognostic studies and anthelminthic activity of A. propinquum Schott. Data composed from such studies can be used as a standard in the quality control of this plant used as an herbal medicine for the treatment of various diseases.


SUMMARY
• In the present era, the use of herbal products and herbal formulations are predominantly increased due to their least side effect and tremendous level of active ingredients. Till date, no detailed standardized work has been reported for this plant. The pharmacognostical parameters such as acid values, extractive values, foaming index, swelling index, fluorescence analysis, and preliminary phytochemical screening provide significant information by which one can easily authenticate the crude drug and also check the adulteration for the quality of raw material. The pharmacognostic parameters studied during the present research work are being reported for the first time and will be quite beneficial for the identification of herbal plant and also for the preparation of herbal monographs. The present study will also be helpful in differentiating the present species from its closely related species of the same genus and family. • Indian adult earthworms (Pheretima posthuma) were used for studying Anthelminthic activity of the herbal drugs in the present study as they share similar anatomical and physiological resemblance with the helminthic parasites of humans. In the present work, both methanolic and aqueous extracts of A. propinquum Schott showed good anthelmintic activity in a dosage-dependent manner as compared to aqueous and standard drug albendazole.

INTRODUCTION
The genus Arisaema, commonly known as cobra lilies, comprised more than 399 herbaceous species belonging to family Araceae. Arisaema propinquum Schott is commonly known as Wallich's cobra lily or cobra lily, and in Kashmir, the plant is known as Hapat-makei or Hapat-mundh. A. propinquum Schott is a tuberous plant that grows at an altitude of 2400-3600 m. The plant is widely distributed throughout temperate to tropical areas, mainly distributed in the Himalayas, from Kashmir to
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Southeast Tibet. [1] The plant grows up to 5-6 feet in height with three large yellowish-green glossy leaves. The spathe or hood is thick at the base and ends in a pointy beak, which comprised 8-20 cm wipe such as long tongue emerging out from inside. At the curvature of the hood, there is a white netting effect which presents the side edges such as a stained window. Leaf stalk and stem are often brown-spotted. The plant flowers in the month of May-June. [2] Traditionally, the dried leaves, fruits, and roots are used to treat diabetic neuropathy, rheumatoid arthritis, [3] chronic boils, and skin eruptions. [4] Pastes prepared from the roots were applied externally for the treatment of erysipelas and scabies. [5] Helminthiasis or worm infection is among the most prevalent infections, effecting billions of people throughout the world. [6] The disease spreads by contamination or inadequate sanitation. [7] The helminthic disease decreases the immune responses to various pathogens and leads to serious clinical complications such as undernourishment, anemia, eosinophilia, pneumonia, [8] malaria, tuberculosis, and human immunodeficiency virus. [9] Due to the resistance of gastrointestinal helminthes to various available anthelminthic drugs, the demand for natural anthelmintics is increasing extremely day by day. [10,11] Indian adult earthworms (Pheretima posthuma) were used for studying anthelminthic activity of the herbal drugs in the present study as they share similar anatomical and physiological resemblance with the helminthic parasites of humans.

Collection of plant material and authentication
The rhizomes of A. propinquum Schott were collected from Doodhpathri area of Budgam district at 2730 m in the month of July 2013. The plant was identified and authenticated by Dr. Akhtar H. Malik, Taxonomist, Centre for Biodiversity and Taxonomy, Department of Botany, University of Kashmir, with reference number 1896-KASH.

Chemicals and reagents
All the chemicals used were of analytical grade and were procured from registered dealers such as HiMedia Laboratories Pvt. Ltd., Mumbai, and Central Drug House Ltd., New Delhi, India.

Macroscopical and Microscopical Evaluation
Macroscopical and microscopical evaluations of A. propinquum Schott were studied according to the methods described in Trease and Evans Pharmacognosy. [12,13] The study was carried out to prevent adulteration which mainly occurs during the selection procedures of raw medicinal material from genus Arisaema.

Preparation of extracts
The fresh air-dried rhizomes of A. propinquum Schott was powdered and then subjected to hot extraction using methanol as a solvent in a Soxhlet apparatus. Aqueous extract was prepared by the decoction method. The extracts obtained were filtered through Whatman filter paper and concentrated under reduced pressure using a rotary vacuum evaporator. The extracts were then dried and transferred in a closed airtight container for further use.

Physiochemical analysis
The physicochemical analysis of A. propinquum Schott rhizomes was carried out by determining ash values, extractive values, foreign organic matter, moisture content, and pH of different solvents. [14] Other parameters such as swelling index, foaming index, [15] total tannin content, and total fat content [16] were also determined.

Fluorescence analysis
Many herbs show fluorescence when cut surface or powder is exposed to ultraviolet (UV) light, and this can be useful in their identification. The fluorescence character of the powdered plant parts (40 mesh) was studied both in daylight and UV light (254 and 366 nm) and after treatment with different reagents such as sodium hydroxide, picric acid, acetic acid, hydrochloric acid, nitric acid, iodine, and ferric chloride. [17,18]

Phytochemical investigation
Weighed quantity of plant material was extracted separately with petroleum ether, chloroform, ethyl acetate, methanol, and water by hot extraction method. The extracts obtained were subjected to preliminary phytochemical investigation for the detection of following compounds: carbohydrates, protein, amino acids, fats and oils, sterols and steroids, glycoside, coumarins, flavonoids, alkaloids, tannins and phenolic compounds, acidic compounds, saponins, terpenes and terpenoids, mucilage resins, and lipids/fats. [19,20] Heavy metal analysis Wet digestion procedure 1.0 g of plant material was digested with 5 mL mixture of nitric and perchloric acids in a digestion flask. The mixture was heated at 110°C until the solution became clear, indicating the complete digestion of the organic matter. The mixture was allowed to cool at room temperature. The digested solution was then diluted with 100 mL of deionized water and was analyzed using an atomic absorption spectrometer (AAS) for the determination of lead, copper, cadmium, and chromium. [21] Anthelminthic activity study Earthworms were collected locally from Hazratbal district, Srinagar. Earthworms of 6-8 cm in length and 0.2-0.4 cm in width were collected and washed thoroughly in saline water to remove the external debris to be used for anti-helminthic activity. The earthworms were acclimatized to the laboratory condition before experimentation. The earthworms were divided into 6 groups of 5 earthworms in each and placed in Petri dishes containing 15 mL of sample/drug solutions, as mentioned below: Group 1: Received 2% gum acacia which served as the control Group 2: Received albendazole suspension at a dose of 10 mg/mL which served as the standard Group 3: Received methanolic extract at a dose of 50 mg/mL Group 4: Received methanolic extract at a dose of 100 mg/mL Group 5: Received aqueous extract at a dose of 50 mg/mL Group 6: Received aqueous extract at a dose of 100 mg/mL. Anthelmintic potential of methanolic and aqueous extracts of rhizomes of A. propinquum was carried out using the method previously described by Ajaiyeoba et al. 2001, with necessary modifications. [22] The Indian earthworms (P. posthuma) of nearly equal size were taken in Petri plates containing 15 mL of different concentrations (50 and 100 mg/ mL) of methanolic and aqueous extracts suspended in normal saline. Albendazole suspension of the same concentration prepared in normal saline was taken as standard. All Petri dishes were kept under room temperature and under close observation. Observation was made for time taken to complete paralysis (PT) and death (DT) for individual worms. Time for paralysis was noted either when any movement could not be observed except when the worms were shaken vigorously or when dipped in warm water (50°C). Death was included when the worms lost their motility followed by white secretions and fading away of their body color. [23]

Macroscopic characters
The rhizomes of A. propinquum Schott are spherical in shape with light brown color outside and cream color inside with pungent odor and astringent taste. The outer covering of the rhizomes is smooth and is of varying size ranging from 2 to 6 cm in diameter [ Figure 1].

Microscopic characters
The transverse section (TS) of the rhizomes was circular in outline and showed the outer multilayered cortex cells, parenchymatous cells, schizogenous intercellular cavities, and well-developed xylem and phloem vessels [ Figure 2]. The powdered microscopy of the rhizomes revealed the presence of cork cells, reticulate vessels, and prismatic calcium oxalate crystals [ Figure 3].

Physicochemical analysis
For the evaluation of pharmacognostic characters of A. propinquum Schott rhizomes, various physicochemical parameters were studied. A. propinquum rhizomes showed the presence of total ash -6.32%, acid insoluble ash -1.77%, water-soluble ash -5.15%, sulfated ash -8.55%, moisture content -18.50%, swelling index -25.66%, and foreign organic matter -1.1% [ Table 1]. For determination of the extractive value, the powdered drug was extracted by three methods, namely cold extraction, hot extraction, and successive extraction [ Table 2]. The fluorescence analysis, an important parameter for the determination of quality and purity of powdered drug material, was also evaluated [ Table 3]. The powered drug is also tested for heavy metal analysis using AAS [ Table 4].    compared to standard albendazole. However, methanolic extract showed potent activity as compared to aqueous extract [ Table 6 and Figure 4].

Statistical analysis
Results are expressed as mean ± standard error mean (n = 6), and the comparisons are made by ANOVA followed by Tukey's test. GraphPad Prism 6 (GraphPad Software, San Diego, California) was used for the analyses. The present endeavor of the study is to establish, as possible, the diagnostic characteristics of A. propinquum Schott rhizomes. In this regard, the macro-and microscopic study was carried out, showed the rhizomes are spherical in shape with light brown color outside and cream color inside with pungent odor and astringent taste. The TS of the rhizome showed the multilayered cortex cells, parenchymatous cells, schizogenous intercellular cavities, and well-developed xylem and phloem vessels. Physiochemical analysis showed 6.32% total ash, 1.77% acid insoluble ash, 5.15% water-soluble ash, and 8.55% sulfated ash. Extractive values, which are important parameters for the evaluation of crude drugs, determine the amount and nature of chemical constituents present within the crude drug. The pH value provides information about the acidic or basic nature of constituents present in the crude drug. For quality assurance, it is necessary to perform heavy metal analysis of the crude drug which was carried out according to the wet digestion method, and the results showed the absence of heavy metals in the crude drug indicating their safety for human consumption. The phytochemical analysis of the extracts showed the presence of alkaloids, carbohydrates, cardiac glycosides, coumarins, proteins and amino acids, phenols, tannins, flavonoids, saponins, steroids, and terpenoids. Anthelminthic activity of methanolic and aqueous extracts was evaluated against Indian adult worm (P. posthuma). Both methanolic and aqueous extracts of A. propinquum Schott rhizomes showed good anthelmintic activity in a dosage-dependent manner. Methanol extract showed the highest anthelmintic activity as compared to aqueous and standard drug albendazole.

CONCLUSION
This study revealed that methanolic and aqueous extracts of A. propinquum Schott rhizomes possess good anthelminthic activity. Therefore, the extracts mainly methanolic which shows the highest activity has the potential for development as an anthelminthic agent against helminthic diseases which however needs further study to understand the underlying mechanism of extracts as an anthelminthic agent. Also, the data generated from the pharmacognostic and phytochemical analysis can be used as a standard in the quality control of this plant.

Phytochemical analysis
The results of qualitative phytochemical analysis of A. propinquum Schott rhizomes are shown in Table 5. The rhizomes showed the presence of alkaloids, carbohydrates, cardiac glycosides, coumarins, proteins, amino acids, phenols, tannins, flavonoids, saponins, steroids, and terpenoids. The anthelminthic activity of methanolic and aqueous extracts of A. propinquum Schott was evaluated against Indian adult earthworms (P. posthuma) due to its similar anatomical and physiological resemblance with the intestinal worms. Methanolic and aqueous extracts of A. propinquum Schott showed dose-dependent anthelminthic activity as