Formulation and Phytochemical Standardization of Achyranthes Aspera L

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International Journal of Pharmaceutical Science & Innovation Volume 1 Issue 2 Formulation and Phytochemical Standardization of Achyranthes Aspera L.

Deepesh Chouhan1, Mani Singh Jadaun2, Toni Shah3 1Vice-Principal, 2,3Lecturer Dr.Tandon Pharmacy College, Agra, Uttar Pradesh, India *Corresponding Authors Email Id: [email protected]; [email protected]; [email protected]

ABSTRACT The aim of the present work was to Formulate & phytochemical standardization of Achyranthes aspera (L.). In order to investigate the traditional, folkloric and ethanomedicinal uses of the plant, the present study was undertaken on the basis of literature review, the work was planned to examine the anti-bacterial activity of extract of leaves of Achyranthes aspera (L.)The preliminary phytochemical screening was also undertaken and it revealed that the plant’s aqueous extracts contains triterpenoid saponins which possess oleanolic acid as the aglycone. Ecdysterone, an insect moulting hormone, and long chain alcohols are also found in Achyranthes aspera. Other chemical constituents such as achyranthine, betaine, pentatriacontane, 6-pentatriacontanone, hexatriacontane, and tritria -contane are also present. Achyranthes aspera (Amaranthaceae) is an important medicinal herb found as a weed throughout India. Though almost all of its parts are used in traditional systems of medicines, seeds, roots and shoots are the most important parts which are used medicinally. The present article gives an account of updated information on its phytochemical and pharmacological properties. The review reveals that wide numbers of phytochemical constituents have been isolated from the plant which possesses activities like antiperiodic, diuretic, purgative, laxative, antiasthmatic, hepatoprotective, anti-allergic and various other important medicinal properties. The crushed plant is used in pneumonia and infusion of the root is used as mild astringent in bowel complaints. Decoction of powdered leaves with honey or sugar candy is useful in early stages of diarrhoea and dysentery. For the last few decades or so, extensive research work has been done to prove its biological activities and pharmacology of its extracts. Saponins, oleonolic acid, dihydroxy ketones, alkaloids, long chain compounds and many other chemical constituents have been isolated.

Keywords: Achyranthes aspera, Latjeera, Medicinal properties, chemical constituents, pharmacological activities.

INTRODUCTION Order: Caryophyllales Family: Amaranthaceae Family: Amaranthaceae Botanical Name: Achyranthes aspera Genus: Achyranthes Common Name: Aandhijhara Species: aspera

CLASSIFICATION Part Used: Whole plant, specially leaves, Kingdom: Plantae stem. Subkingdom: Tracheobionta Division: Magnoliophyta Medicinal Properties: Anti- Class: Magnoliopsida inflammatory, Expectorant, astringent, Subclass: Caryophyllidae Alexiteric, Appetiser, Stomachic,

International Journal of Pharmaceutical Science & Innovation Volume 1 Issue 2 Deobstruent, Antispasmodic, Antiseptic, remedies. It is estimated that about 80% Dentrifrices. of the world population residing in the vast rural areas of the developing and Medicinal Use: Dental Caries, Asthma, under developed countries still depends Itching, Infusion and Hysteria, Externally mainly on medicinal plants. It is quite in skin diseases, in foul ulcers, convulsions obvious that the plant is widely used in in children diseases. The leaves are also traditional medicinal system of India and useful in tooth decay. has been reported to possess hepatoprotective, anti-inflammatory, Chemistry: leaves contains: triterpenoid antitussive, antifungal and also used to saponins, betaine, achyranthine, check wounds healing and antibacterial hentriacontane, ecdysterone and two properties. Achyranthes aspera is a very glycosides of oleanolic acid. Seeds: amino important plant for its large number of acids. medicinal properties as well as medicinally important chemicals like ecdysterone, Regional Habitat: Found in tropical areas achyranthine, betaine, pentatriaontane, 6- pentatriacontanone, hexatriacontane and Description: It is an erect or prostrate, tritriacontane. The plant shows many annual or perennial herb about 1m to 2m pharmacological activities like, anti- high. Leaves are opposite, margins allergic, cardiovascular, nephroprotective, undulate. Flowers: numerous, stiffly antiparasitic, hypoglyceamic, analgesic deflected against the pubescent rachis in and antipyretic. Many traditional uses are elongate terminal spike, 20-30cm. long. also reported like antiperiodic,purgative Seeds are oblong-ovoid [1]. and laxative, in various types of gastric disorders and in body pain which are being studied till today and further research has to be done. Achyranthes aspera L., locally known as “Telenge or ambulale” is one of the traditionally used anti-fertility plants in the indigenous health care delivery system of Ethiopia. It is a stiff erect perennial herb of 1–3 feet with simple elliptic leaves. The extracts of leaves, roots, and seeds of the plant have been used for control of fertility, in placental retention, and in postpartum bleeding. The preliminary study on leaves extract of the plant had shown some antifertility effect. The present study was Fig. 1. Archyranthes aspera L. leaves investigated the effect of methanolic leaves extract on fetal abortion, uterine and Plant Achyranthes aspera L [2] pituitary weights, gonadal horomones, Achyranthes aspera is a common plant serum lipids and in female rats in attempt found throughout India this review focuses to further validate scientifically. The on the wide pharmacological activities of traditional claim a perennial stiff erect Achyranthes aspera L. Before the herb, 2.0 m high is growing up to 1000 m introduction of modern medicines, disease height. Stems are square, leaves elliptic treatment was entirely managed by herbal ovate or broadly rhombate, 5-22 cm long,

International Journal of Pharmaceutical Science & Innovation Volume 1 Issue 2 2.5 cm broad, and adpressed pubescent. addition of Achyranthes aspera would The inflorescences are 8 - 30 cm long, enhance the efficacy of any drug of plant with many single, white or red flowers, 3 - origin [3]. 7mm wide. Flowering time is in summer. The plant is widespread in the world as a Morphological Characters of weed, in Baluchistan, Ceylon, Tropical Achyranthes Aspera L Asia, Africa, Australia and America. In the Achyranthes aspera L locally is one of the northern part of India it is known as a most important traditionally used medicinal plant in different systems of antifertility plants in the indigenous health folk medicine. Achyranthes aspera L, a care delivery system of Ethiopia. It is a stiff erect herb, has been reported to stiff erect perennial herb of 1 to 3 feet with posse’s medicinal properties. simple elliptic leaves. The ex-tracts of leaves, roots, and seeds of the plant have According to World Health been used for control of fertility, in Organization, medicinal plants are the placental retention, and in postpartum best source to obtain a variety of newer bleeding. The preliminary study on leaves herbal drugs. About 80% of individuals extract of the plant had shown some from developed countries use traditional antifertility effect. Flowering time of this medicine, which has compounds derived plant is in summer. The stems are square, from medicinal plants. Therefore, such leaves elliptic ovate or broadly rhombate. plants should be investigated to better The inflorescences are 8 - 30 cm long, understand their properties, safety and with many single, white or red flowers,3 - efficacy. The use of plant extracts and 7mm wide .The plants is widespread in the phytochemicals, both with known world as a weed, in Baluchistan, Ceylon, antimicrobial properties, can be of great Tropical Asia, Africa, Australia and significance in therapeutic treatments. In America. In the northern part of India it is the last few years, a number of studies known as a medicinal plant in different have been conducted in different countries systems of folk medicine [4]. to prove such efficiency. Many plants have been used because of their antimicrobial Traditional Uses traits, which are chiefly due to synthesized Traditionally, the plant is used in asthma during secondary metabolism of the plant. and cough. It is pungent, antiphlegmatic, antiperiodic, diuretic, purgative and Achyranthes aspera L. (Family laxative, useful in oedema, dropsy and Amaranthaceae) is a common plant of the piles, boils and eruptions of skin etc. study area abundantly found in wastelands. Crushed plant is boiled in water and is It is known as “Prickly chaff flower” in used in pneumonia. Infusion of the root is English and “Chirchita”, “Onga”, a mild astringent in bowel complaints. The “Latjeera” or “Apamarga” in local flowering spikes or seeds, ground and language and dialects. The plant is highly made into a paste with water, are used as esteemed by traditional healers and used in external application for bites of poisonous treatment of asthma, bleeding, in snakes and reptiles, used in night blindness facilitating delivery, boils, cold, cough, and cutaneous diseases. For snake bites the colic, debility, dropsy, dog bite, dysentery, ground root is given with water until the ear complications, headache, leucoderma, patient vomits and regains consciousness. pneumonia, renal complications, scorpion Inhaling the fume of Achyranthes aspera bite, snake bite and skin diseases mixed with Smilax ovalifolia roots is etc…Traditional healers claim that suggested to improve appetite and to cure

International Journal of Pharmaceutical Science & Innovation Volume 1 Issue 2 various types of gastric disorders . It is of the isolated compound was established useful in haemorrhoids, leaves and seeds by modern spectroscopic techniques [6]. are emetic, hydrophobia, carminative, resolve swelling, digestive and expel Pharmacological Actions [6,7] phlegm. Ash of the plant is applied 1) Hepatoprotective – Apamarg shows externally for ulcers and warts. The hepatoprotective activity and decreases crushed leaves rubbed on aching back to the levels of SGPT, SGOT, Alkaline cure strained back . A fresh piece of root is Phosphatase and total bilirubin.. used as tooth brush. Paste of the roots in 2) Diuretic – Apamarg significantly water is used in ophthalmia and opacities increases renal clearance of sodium, of the cornea. Paste of fresh leaves is used potassium and chloride ions due to its for allaying pain from bite of wasps. The diuretic property. plant is useful in liver complaints, 3) Immunostimulant – A. aspera has rheumatism, scabies and other skin immunostimulant property and it diseases. It also possesses tranquillizing stimulates cell mediated immune properties [5]. system by increasing phagocytic function. Chemical Constituents 4) Antidiabetic – A. aspera helps to 63.05% of the oil was identified. reduce the blood glucose level to its Hydroquinone (57.7%) was found to be normal by increasing plasma insulin. the Chief Constituent. Preliminary 5) Antioxidant- Apamarg, effectively chemical examination of the seeds of prevents free radical mediated cell Achyranthes aspera which were damage by free radicals scavenging identified.asα-L-rhamnopyranosyl-(1_4)- activity and thus can be used as a (β- Dglucopyranosyluronicacid)-(1_3)- potent source of natural antioxidant oleanolic acid, α-L rhamnopyranosyl- compounds. Hence, it is very useful in (1_4)-(β- Dglucopyranosyluronicacid)- diseases like diabetes, high cholesterol (1_3)-oleanolic acid-28-O-β-D- etc. glucopyranoside and α- 6) Cardiovascular Activity - Lrhamnopyranosyl-(1_4)-(β- Achyranthine, a water-soluble alkaloid Dglucopyranosyluronic acid)-(1_3)- isolated from Achyranthes aspera, oleanolic acid-28-O-β-Dglucopyranosyl- decreases blood pressure and heart (1_4)-β-Dglucopyranoside.6 Betaine, rate, dilates blood vessels, and achyranthine, hentriacontane, ecdysterone, increases the rate and amplitude of achyranthes saponins A,B,C,D are the respiration. major chemical constituents found in Apamarg. [7] R.D. Rameshwar (2007) STANDARDIZATION AND QUALITY isolated chemical compounds of the CONTROL OF HERBAL CRUDE volatile oil from Achyranthes aspera DRUGS– PROCESSES AND leaves, growing in Dehra Dun were PROCEDURES [8] analyzed by G.C. M.S. Seven compounds According to WHO, standardization and viz., pbenzoquinone, hydroquinone, quality control of herbals is the process spathulenol, nerol, α-ionone, asarone and involved in the physicochemical eugenol constituting Aziz. et. al. (2005), evaluation of crude drug covering aspects, isolated 3-Acetoxy-6-benzoyloxyapang - such as selection and handling of crude amide from an ethyl acetate extract of the material, safety, efficacy and stability stem of Achyranthes aspera. The structure assessment of finished product, documentation of safety and risk based on

International Journal of Pharmaceutical Science & Innovation Volume 1 Issue 2 experience, provision of product 10) Toxicological studies: This helps to information to consumer and product determine the pesticide residues, promotion. Attention is normally paid to potentially toxic elements, safety such quality indices such as: studies in animals like LD50 and Microbial assay to establish the 1) Macro and microscopic examination: absence or presence of potentially For Identification of right variety and harmful microorganisms. search of adulterants. 2) Foreign organic matter: This involves Physical Evaluation [8] removal of matter other than source Each monograph contains detailed plant to get the drug in pure form. botanical, macro-scopic and microscopic 3) Ash values: These are criteria to judge descriptions of the physical characteristics the identity and purity of crude drug – of each plant that can be used to ensure Total ash, sulphated ash, water soluble both identity and purity. Each description ash and acid insoluble ash etc. is accompanied by detailed illustrations 4) Moisture content: Checking moisture and photographic images which provide content helps reduce errors in the visual documentation of accurately estimation of the actual weight of drug identified material. material. Low moisture suggests better stability against degradation of Microscopic Evaluation [7,8] product. Full and accurate characterization of plant 5) Extractive values: These are indicative material requires a thorough physical weights of the extractable chemical examination. Microscopic analyses of constituents of crude drug under plants are invaluable for assuring the different solvents environment. identity of the material and as an initial 6) Crude fibre: This helps to determine screening test for impurities. the woody material component, and it is a criterion for judging purity. Chemical Evaluation [5,8] 7) Qualitative chemical evaluation: This This covers screening, isolation, covers identification and identification and purification of the characterization of crude drug with chemical components. Chemical analysis respect to phytochemical constituent. It of the drug is done to assess the potency of employs different analytical technique vegetable material in terms of its active to detect and isolate the active principles. The chemical screening or tests constituents. Phytochemical screening may include colour reaction test, which techniques involve botanical help to determine the identity of the drug identification, extraction with suitable substance and possible adulteration. solvents, purification, and characterization of the active Biological Evaluation [5,8] constituents of pharmaceutical Pharmacological activity of certain drugs importance. has been applied to evaluate and 8) Chromatographic examination: Include standardize them. identification of crude drug based on the use of major chemical constituents The assays on living animal and on their as markers. intact or isolated organs can indicate the 9) Quantitative chemical evaluation: To strength of the drug or their preparations. estimate the amount of the major These assays are known as Biological classes of constituents. assays or Bioassay.

International Journal of Pharmaceutical Science & Innovation Volume 1 Issue 2 Purity Determination agent, it is in accordance with its use in Each monograph includes standards for traditional medicine. purity and other qualitative indices already 5) Medicinal Properties of Achyranthes mentioned above. aspera, by Ashwini Kumar Sharma, in International Journal of Ayurveda & Analytical Methods Pharma Research. Critical to compliance with any 6) Achyranthes aspera- a potent monograph standard is the need for immunostimulating plant for appropriate analytical methods for traditional medicine by Pravin Kumar determining identity, quality, and relative Shrivastava, in International Journal of potency. There are a plethora of analytical Pharmaceutical Sciences & Research. methods available. However, it is often 7) Phytochemical & Pharmacognostical difficult to know which is the most Profile of Achyranthes aspera, by appropriate to use, but critical among Anand Kumar and Koushleh k. Mishra, know analytical tools in monograph in Journal of Pharmacognosy And standardization is chromatography [9]. Phytochemistry 8) Ethnomedical Uses of Achyranthes REVIEW OF LITERATURE aspera L., in Orissa (India), by R.D. 1) Phytochemical Screening and GCMS Girach, Aminuddin and S.A. Khan, in analysis of Achyranthes aspera Linn. International Journal of By T.P Kumari Pushpa, A Doss in Pharmacognosy, 1992. International Journal of Chemistry 9) Kumar A. et. al., evaluate the in-vitro Studies. Streptococcus mutans activity of 2) Phytochemical constituents and Vaishvanara Churna. Vaishvanara Pharmacological uses of medicinal churna is a ayurvedic formulation used plants Achyranthes aspera by traditionally as antimicrobial analgesic, Janmayjoy Banerjee, published in anti-inflammatory agent. Vaishvanara world journal of pharmaceutical Churna composed of Saindhava reseach, 2014 lavana, Ajowan, Ajamoda, Shunti, and 3) Yadav R. D. et. al., reviewed the Haritaki. Effect of aqueous extract of herbal plants used in the treatment of aspera on calcium oxalate Tooth caries, the present review aims crystallization was evaluated. to give data highlighting the present 10) Antimicrobial Activity of Gokhsuradi trends in research of medicinal plants Churan, was studied by Srinivasa accredited with antimicrobial activity. A.K.B. et. al., The study suggested 4) The inhibition of in-vitro Achyranthes aspera has a potential Streptococcus mutan, by Achyranthes inhibition of microbes of Strptococcus aspera L. extract was investigated by 11) Pharmacognostic Evaluation of Agarwal K. K. et. al., by different different parts of Achyranthes aspera methods, the aim was to evaluate the Linn., by Rupesh k. Singhal, D.C. effectiveness of different Singh, A.K. Gakkhar, Suresh Chaube, concentrations of the extract. Thus, in International Journal Of Ayurveda Acyranthes aspera L. was found to be & Pharma Research. a potent, promising antimicrobial 12) Agrawal K.K. et. al., was studied the

International Journal of Pharmaceutical Science & Innovation Volume 1 Issue 2 In-vitro Calcium Oxalate evaluated. Crystallization Inhibition by 19) Phytochemical Screening and GCMS 13) Achyranthes aspera L. and analysis of Achyranthes aspera Linn. Bryophyllum pinnatum Lam. In the By T.P Kumari Pushpa, A Doss in nucleation assay the% inhibition for International Journal of chemistry calcium oxalate crystal formation was studies. found to be directly proportional to the 20) Phytochemical constituents and increase in concentration of the plant Pharmacological uses of medicinal extracts. Achyranthes aspera showed plants Achyranthes aspera by maximum inhibition of 60.06±0.19% Janmayjoy Banerjee, published in at 1000 mg/ml while Bryophyllum world journal of pharmaceutical pinnatum showed maximum inhibition reseach, 2014 of 49.93±0.07% at the same 21) Yadav R. D. et. al., reviewed the concentration. herbal plants used in the treatment of 14) Medicinal Properties of Achyranthes Tooth caries, the present review aims aspera, by Ashwini Kumar Sharma, in to give data highlighting the present International Journal of Ayurveda & trends in research of medicinal plants . Pharma Research. 22) The inhibition of in-vitro 15) Achyranthes aspera- a potent Streptococcus mutan, by Achyranthes immunostimulating plant for aspera L. extract was investigated by traditional medicine by Pravin Kumar Agarwal K. K. et. al., by different Shrivastava, in International Journal of methods, the aim was to evaluate the Pharmaceutical Sciences & Reseach. effectiveness of different 16) Phytochemical & Pharmacognostical concentrations of the extract. Thus, Profile of Achyranthes aspera, by Acyranthes aspera L. was found to be Anand Kumar and Koushleh K. a potent antimicrobial agent, it is in Mishra, in Journal of Pharmacognosy accordance with its use in traditional And Phytochemistry medicine. 17) Ethnomedical Uses of Achyranthes 23) Medicinal Properties of Achyranthes aspera L., in Orissa (India), by R.D. aspera, by Ashwini Kumar Sharma, in Girach, Aminuddin and S.A. Khan, in International Journal of Ayurveda & International Journal of Pharma Research. Pharmacognosy, 1992 24) Achyranthes aspera- a potent immune- 18) Kumar A. et. al., evaluate the in-vitro stimulating plant for traditional Streptococcus mutans activity of medicine by Pravin Kumar Vaishvanara Churna. Vaishvanara Shrivastava, in International Journal of churna is a ayurvedic formulation used Pharmaceutical Sciences & Research. traditionally as antimicrobial analgesic, 25) Pharmacognostic Evaluation of anti-inflammatory agent. Vaishvanara different parts of Achyranthes aspera churna composed of Saindhava lavana, Linn., by Rupesh k. Singhal, D.C. Ajowan, Ajamoda, Shunti, and Singh, A.K. Gakkhar, Suresh Chaube, Haritaki. Effect of aqueous extract on in International Journal of Ayurveda & calcium oxalate crystallization was Pharma Research.

International Journal of Pharmaceutical Science & Innovation Volume 1 Issue 2 EXPERIMENTAL METHODOLOGY Collection and Preparation of extract of Achyranthes aspera L.

Preliminary Phytochemical Screening of the Extract Physical Evaluation The dried leaves of Achyranthes aspera L. 1) Determination of Moisture Content: were subjected to qualitative screening for 2 g of each sample were placed in pre- the various phytoconstituents. Tests for weighed flat porcelain dish. Dry in the common phytochemicals were carried out oven at 100oC±5oC till the constant by standard methods. Following methods weight was obtained. The loss of are used for phytochemical screening of weight was calculated with reference different extract mentioned by Kulkarni to air dried material. (2005) [10]. 2) Determination of Total Ash Content: 2 gm of air dried powder was placed as Preparation of Extract by Sequential a uniform layer in crucible silica and Extraction Method ignite gradually up to 500600oC until 1) Preparation of Methanolic Extract: it was white indicating the absence of The different parts of plant were shade carbon, allowed to cool and weighed to dried and extract was prepared in determine the percentage of ash with methanol by filtering the mixture at reference to air-dried respective regular intervals. samples. 2) Preparation of Aqueous Extracts: 3) Determination of Acid Insoluble Ash The filtrate of methanolic extract was Content: The ash was boiled with shade dried and with this filtrate the dilute HCL for 5 minutes and insoluble aqueous extract is prepared in distilled matter was collected in a sintered glass water. crucible washed, ignited, and cooled

International Journal of Pharmaceutical Science & Innovation Volume 1 Issue 2 finally it was weighed to calculate the obtained indicating the presence of percentage of acid-insoluble ash with proteins in it. reference to the bone dried material. 4) Determination of Water Soluble Ash OBSERVATIONS AND RESULTS Content: Total ash was boiled with Physicochemical Parameters of water for 5 minutes and insoluble ash Achyranthes aspera: In physicochemical was collected in a sintered glass parameters moisture content, total ash crucible washed ignited at a content, acid soluble ash content, water temperature not exceeding 450oC. soluble ash content and solvent extractive 5) Cool and weighed for the value was observed. In A. aspera extract, determination of water soluble ash the moisture content in stem and roots with reference to the bone dried drug. (40%) were more than the values in leaf 6) Determination of Solvent Extractive and inflorescence, total ash content in stem Values: 5gm of the air dried, (7%) was more followed by leaf then powdered macerated with 100 ml of inflorescence and least in roots, acid solvent for 24 hours, shaken frequently insoluble ash content was found highest in and allowed to stand for 24 hours. stem and inflorescence (1%) then in leaf Thereafter, filtered, evaporated the and least in roots and water soluble ash filtrate to dried and weight was taken. content was found highest in inflorescence The percentage of solvent soluble (5.5%) followed by stem and leaves. extractive with reference to bone dried sample has to be calculated. Parameters Values (%) w/w Moisture % Total Ash content Acid Insoluble Ash Chemical Evaluation content Water Soluble Ash content Leaf Test for Saponins 30% 5% 0.98% 4.0% Stem 40% 7% Foam test: Samples were dissolved in 1.00% 5.0%. distill water and shaken vigorously. A layer of foam on top layer was formed We observe the highest percentage yield which is stable, indicates the presence of for methanol extract was found in leaf saponins in the sample. extract (7% and 8%) while for aqueous it was (10%) and least for both solvents was Test for Flavonoids for roots. The color observed as green for NaOH Test: Taken 1ml of the sample with methanolic leaf and stem extract. While 10ml of 1% NaOH solution and gently for aqueous extract it was dark brown for shaken the sample, yellow color was leaf and stem extract. observed denoting the presence of flavonoids. In-Vitro Testing of Achyranthes Aspera on Streptococcus Mutans Test for Glycosides Dental caries and periodontal diseases Hansch Test: In aqueous extract conc. have historically been considered the most H2SO4 was added from the side walls and important global oral health burdens. formation of a brown ring suggested the Many chemicals and synthetic drugs have presence of carbohydrates. proven to be effective in the prevention of these diseases, but they also have marked Test for Proteins side effects. In recent times, there has been Xanthoprotein Test: Mix 3 ml extract a marked shift toward herbal cures because solution with 1 ml conc. H2SO4 and of the pronounced cumulative and boiled it by which yellow precipitate was irreversible reactions of modern drugs.

International Journal of Pharmaceutical Science & Innovation Volume 1 Issue 2 of S. mutans (Microbiological Type Achyranthes aspera Linn. is a species of Culture Collection [MTCC] 890) were plant in the Amaranthaceae family, obtained from MTCC, Institute of commonly known Microbial Technology, Chandigarh. as Apamarga or Chirchita in Hindi. It is a popular medicinal plant which has Preparation of Achyranthes aspera occupied a pivotal position in folk Extract medicine throughout the tropical Asian Fresh stems and leaves of A. aspera were and African countries. It is distributed collected and dried under shade and then throughout the tropical world and it is in an oven at a temperature <50°C. The commonly found as a weed throughout stems and leaves were ground into a fine India. powder. Then, 100 g of powdered stems and leaves were boiled in 2000 ml of This herb is reported to have various distilled water separately in two flasks for pharmacological actions such as anti- 2 h. One thousand and three hundred inflammatory, analgesic, and antipyretic milliliters of filtrate was obtained from the activities. It is used to treat many problems stems and leaves, which were then filtered such as piles, digestive disorders, fever, using a filter paper. It was reduced to cough, dysentery, psoriasis, paralysis, obtain a solid residue of 6 g by heating it spleen enlargement, control of fertility, at 60°. The solid residue obtained was then and postpartum bleeding. While some stored at a low temperature. Dimethyl people also use the roots of this plant as sulphoxide was used to dissolve the solid tooth brush, it still has a long way to go to residue made from the stems and leaves to be established as a recognized entity in the make different concentrations. One gram prevention and treatment of oral diseases. of solid residue was mixed in 10 ml of dimethyl sulphoxide to make 10% Various studies report that the aqueous concentration and it was kept as a stock solution of A. aspera has shown solution. From this stock solution, further antibacterial activity against different concentrations were made. Staphylococcus aureus, Streptococcus heamolyticus, and Bacillus typhosus, while Culture Media alcoholic and aqueous extract of its leaves Brain heart infusion agar was used as a possess antibacterial activity against S. medium to grow the lyophilized bacteria. aureus and Escherichia coli. Very few The bacteria were streaked to the agar studies have been conducted on the slants in test tubes and were kept in an antibacterial effect of A. aspera extracts incubator at 37°C for 48 h. Cup plate against oral pathogens. Therefore, the aim method was used to determine the zones of of this study is to evaluate the in inhibition. In this method, the circular vitro antibacterial activity of A. wells that can incorporate different aspera against Streptococcus mutans. volumes of the test agent were cut in the agar plates using a template. The extract of MATERIALS AND METHODS stems and roots with different About the Plant and Microorganism concentrations (0.5%, 1%, 1.5%, 2%, The A. aspera plant was collected from the 2.5%, 5%, 7.5%, and 10%) and at different outskirts of Gurgaon, Haryana, and was volumes was transferred to the agar plates. authenticated by an expert, National About 0.2% chlorhexidine at 10 µl was Research Centre on Plant Biotechnology, used as control and it was also transferred Pusa Campus, New Delhi. Pure strains to agar plates which were incubated

International Journal of Pharmaceutical Science & Innovation Volume 1 Issue 2 aerobically at 37°C for 24 h. Antibacterial Figures 1 shows the zones of inhibition activity was interpreted from the size of formed by the stem and leaves extracts the diameter of zones of inhibition of A. aspera and chlorhexidine, measured in millimeter using a measuring respectively. guide in all the agar plates.

Fig.1. Zone of inhibition formed by Achyranthes aspera leaf extracts at different concentrations and volumes

Fig. 2. Zone of inhibition formed by Achyranthes aspera leaf extracts at different concentrations & volumes.

International Journal of Pharmaceutical Science & Innovation Volume 1 Issue 2

Fig. 3. Zone of inhibition formed by 0.2% chlorhexidine at 10 μl volume.

Introduction to Herbal Toothpaste extracts to enhance your breath. Contains Toothpaste is a paste or gel dentifrice used calcium carbonate to whiten teeth [12]. with a toothbrush as an accessory to clean and maintain the aesthetics and health of Ingredients used in Herbal Toothpaste teeth. Toothpaste is used to promote oral Calcium carbonate: Mild abrasive and hygiene: it serves as an abrasive that aids polisher. in removing dental plaque and food from the teeth, assists in suppressing halitosis, Titanium Dioxide: Abrasive. and delivers active ingredients (most commonly fluoride) to help prevent tooth Aloe vera: Vegetable-based sweetener and decay (dental caries) and gum disease moistener. (gingivitis). Salt and sodium bicarbonate (baking soda) are among materials that can Sodium lauryl sulphate: Coconut-based be substituted for commercial toothpaste. foaming agent. Toothpaste is not intended to be swallowed due to the fluoride content, but Silica: Mild abrasive and thickener. is generally not very harmful if accidentally swallowed in small amounts; Methyl Cellulose: Gumming agent however, one should seek medical sourced from plant fibre. attention after swallowing abnormally large amounts [11]. Clove oil

Herbal toothpaste Contains Chlorophyll to Menthol help alkaline the mouth. Cleans teeth, freshens breath, and helps prevent tooth Herbal extracts (Achyranthes aspera, decay and the buildup of plaque. Contains babool, Neem) 11 herbal extracts renowned for their cleansing, astringent, anti-microbial, and Sodium Saccharin: Sugar-free sweetener. refreshing properties. Mint and menthol Peppermint oil

International Journal of Pharmaceutical Science & Innovation Volume 1 Issue 2 METHOD OF PREPARATION Extract of Achyranthes aspera Leaves

Extract of Babool Bark

Extract of Neem Bark

International Journal of Pharmaceutical Science & Innovation Volume 1 Issue 2 FORMULATION Formula for 100g of Preparation Ingredients Quantity(gm) Calcium Carbonate 35g Aloe vera 10g Sodium Saccharine 0.40g Titanium Dioxide 0.50g Sodium Lauryl Sulphate 1.50g Benzoic acid 0.1g Menthol 1.0g Clove Oil 0.001g Purified water Q.S

PROCEDURE

Evaluation of Herbal Toothpaste Tube inertness Composition The toothpaste container shall not produce Toothpaste is not composed of mono or any corrosion or deterioration in normal disaccharides such as sucrose or storage conditions like heating temperature fermentable carbohydrates. All ingredients at 45±20C for ten days. Tube inertness can should comply with the Indian standards. be examined by cutting the internal surface, open and observing whether any Homogeneity sign of deterioration or chemical attack The toothpaste shall extrude a occurred in the container. homogenous mass from the collapsible tube or any suitable container by applying Determination of Sharp and Edge of normal force at 27±20C. In addition, Abrasive Particles bulk of contents shall extrude from the Extrude the contents 15-20 cm long on the crimp of container and then rolled it butter paper, repeat the same process for at gradually. least ten collapsible tubes. Press with the

International Journal of Pharmaceutical Science & Innovation Volume 1 Issue 2 contents of the entire length with finger tip for the presence of sharp and hard edged Determination of lead abrasive particles. Toothpaste shall not The color produced with sample solution contain such particles. containing hydrogen sulfide is compared with standard lead solution. Determination of Spreadability One gram of toothpaste placed on a glass Determination of arsenic slide (10 x 10 cm), cover with another Yellow color stains produced when arsine glass slide. Then carefully place two kg gas reacts with mercuric bromide paper. weight of on covered glass slide (sliding, Sample stain is complying with the shall not take place). Measure the standard stain. spreading (in cm) of the toothpaste after 3 minutes. Repeating the experiment and Foaming power note the average value of three readings. Take a suspension of the material in measuring cylinder and shake the Determination of Fineness suspension for 12 times. And measure the Weigh accurately about 10 gm of volume of the foam produced after shaking toothpaste placed in a 100 ml beaker. for 5 minutes. Procedure: weigh 5 g of Allow 50 ml of water, stand for 30 min toothpaste in a 100 ml glass beaker. Add with stirring until the paste gets 10 ml of water, cover the glass beaker with completely dispersed. Transfer the solution a watch glass and stand for 30 minutes. to 150 micron IS sieve and wash with a Heat the suspension gently to dissolve the slow stream of tap water. Allow running detergent if present in it. tap water drained the on sieve and dry (at 105±20C) the sieve by place it in an oven. Stir the suspension with glass rods and Transfer any residue particle is present on transfer it to 250 ml measuring cylinder. the sieve to a watch glass and weighs it. Examine if no foam is produced (more Calculation: (i) Material on the sieve % by than 2 ml). Transfer the residue retained in (Retained mass/Material taken) x 100 (ii) the beaker to measuring cylinder by Weigh accurately about 10 gm of adding of 5- 6 ml of water. Then make up toothpaste placed in a 100 ml beaker. the cylinder with 50ml of water. Stir the Allow 50 ml of water; stand for 30 min contents with up-down movements to get with stirring until the paste gets uniform suspension at 300C. after shaking, completely dispersed. Transfer the solution keep the cylinder stand for 5 minutes. And to 75 micron IS sieve and washes with a final note the volume obtained with foam slow stream of tap water. Allow running + water [13]. tap water drained on the sieve and dry (at 105±20C) the sieve by placing it in an OBSERVATION TABLE oven. Transfer any residue particle is present on the sieve to a watch glass and S.No Parameters Observation weighs it. 1 Colour Slight Brown 2 Odour Characteristic pH determination 3 Taste Sweet Weigh 10 g of toothpaste placed in 150 ml 4 Stability Stable beaker. Allow 10 ml of boiled and then 5 Spreadability Easily Spreadable cooled water. Stir vigorously to make a 6 Abrasiveness Good Abrasive suspension. Measure the pH of the 7 Foamability Good suspension using pH meter. CR Journals (Page 1–18) 2021. All Rights Reserved Page 15

International Journal of Pharmaceutical Science & Innovation Volume 1 Issue 2 RESULTS 10% concentration and 250 µl volume and The lowest concentration of the extract, 21 mm at 10% concentration at 250 µl which inhibited the growth of the tested volume for stem and root extracts, microorganisms, was found to be 2.5% for respectively. Though the plant extract was both the stem and root extracts. The found to be effective at a higher minimum zone of inhibition of 14 mm was concentration and volume than seen at 2.5% concentration and 100 µl chlorhexidine gluconate, it showed a volume of root extract. While the marked antibacterial activity and should be maximum zone of inhibition of 21 mm considered to replace the synthetic drugs was seen at 10% concentration and 250 µl and chemicals because of their irreversible volume. Similarly, the minimum zone of side effects. A study conducted by Giri et inhibition of 12 mm was seen at 2.5% al. showed 28 ± 1.4 zone of inhibition at concentration and 100 µl volume of stem 50% concentration of A. aspera extract. extract while the maximum zone of Similar studies were conducted by inhibition of 16 mm was seen at 10% Kumar et al. and Manjula et al., in which concentration and 250 µl volumes. The the extracts of A. aspera were reported to mean value of zone of inhibition of the have a marked antibacterial activity stem and leaves extracts was 13 and 15.2 against various pathogenic strains. at the lowest volume and 14.7 and 18.5, respectively, at the highest volume. While The cup plate method was used in this for chlorhexidine at 2% concentration, the study which depends on the diffusion of zone of inhibition obtained was 19 mm at the test agent to an extent that growth of 10 µl volume. the added bacteria is prevented completely in a zone around the circular wells DISCUSSION containing the solution of the test agent. Herbal medications have been around for This method has been found to give ages. Our ancestors had been known to use precise results as also reported by the plant life to treat and alleviate illnesses studies of Vahabi et al., Prabhat et al., that affect the human body. Medicinal Phatak et al., and Agarwal et al. plants are today being once again preferred because of the various reasons such as The antibacterial activity of A. aspera can their easy availability, negligible side be attributed to the alkanoids and tannins. effects, low cost of treatment, and their Tannin is a phenolic compound which is effectiveness. The present study proved soluble in water and it could be one of the that A. aspera possesses a significant components responsible for the antibacterial activity against S. mutans, antibacterial activity. which is the causative organism playing a major role in the pathogenesis of dental Extracts of the leaves and callus of this caries [14]. plant in various solvents have been reported to show antimicrobial activity. In the present study, chlorhexidine Prabhat et al. reported that methanolic gluconate which is considered a gold extracts possess antimicrobial activity standard for antibacterial agents against while Khan et al. reported that the ethanol oral pathogens was taken as control. At and chloroform extracts of the seeds of A. 0.2% concentration, it showed a zone of aspera show mild-to-moderate antibiotic inhibition of 19 mm at 10 µl volume. activity against Bacillus subtilis, E. coli, While, for A. aspera, the maximum zone and Pseudomonas aeruginosa. Jebashree of inhibition was found to be 16 mm at et al. demonstrated the anticariogenic

International Journal of Pharmaceutical Science & Innovation Volume 1 Issue 2 activity of A. aspe1ra by using ethyl Hence, by the evidence of in vitro studies, acetate extracts of A. aspera, which it is concluded that Lab made Herbal showed high antibacterial activity toothpaste formulated in a laboratory was against S. mutans than other solvent found to be of good quality. Antibacterial extracts. However, in the present study, agents against oral microorganisms, aqueous extract of A. aspera was used, especially those contributing to sub- and which is most easy and safe to obtain and supra-gingival biofilm formation, play an showed a marked antibacterial activity. important role in the prevention of dental Any antimicrobial agent is considered caries and periodontal disease. Since some effective, given the size of inhibition zone chemical synthetic drugs including produced by it measures 2 mm or more. In chlorhexidine can cause brown staining of the present study, the minimum zones of the teeth, tongue, transient impairment of inhibitions obtained were 12 mm and 14 taste perception toxic effects on connective mm for stem and root extracts of A. tissues, dryness and soreness of oral aspera, respectively. It has proved to have cavity, allergic reactions in patients and potent antibacterial property [15]. oral desquamation in children, herbs with medicinal properties can serve as a useful CONCLUSION and effective source of treatment for The ingredients used in present work, was various dental diseases. Hence, they can be screened and selected to possess the utilized along with conventional medicine antibacterial effect and to maintain oral that can assure us of greater health in the hygiene as it can be claimed by its result as future. Further studies can be conducted in effective tooth paste. Any Herbal future to assess the safety levels of such toothpaste is considered safe to use twice a herbs. daily and it does not cause any harmful effect, instead impart good freshness and REFERENCES away from bad odour. Oral hygine can be 1) Kokate, C.K., Purohit, A.P. and maintained in a reliable safe and Gokhale, S.B., 2005. Pharmacognosy, inexpensive way by using herbal Pune, Nirali Prakashan, pp.1,5, 256, toothpaste. Eventually Herbal toothpastes 276. having an emphasized role in the 2) Khadelwal, K.R., 2008. Hand book of maintaining the oral hygienic nature as practical pharmacognosy, Pune, Nirali well as preventing dental caries. Based on prakashan, pp. 149-156 this pattern, Lab made Herbal toothpaste 3) Dhar ML, Dhar MM, Dhawan BN, was formulated by selecting suitable Mehrotra BN and Ray C: Screening of ingredients to get the formulation more Indian plants for biological activity: stable. Evaluation and comparison of part I”, Ind. J. Exp.Biol. 2008; .6: results with commercial Herbal toothpaste 232-247. are demonstrated that Lab made toothpaste 4) Girach RD and Khan ASA: is having equal patronizing and engrossing Ethnomedicinal uses of Achyranthes passion over the marketed formulations aspera leaves in Orissa, India, Int J (Himalaya, Meswak and Dant Kanti).All Pharmacogn 2011; 30: 113-115. the marketed Herbal tooth pastes and Lab 5) Gokhale AB, Damre AS, Kulkami KR made. This preliminary in vitro study and Saraf MN: Preliminary evaluation demonstrated that Lab made Herbal of anti-inflammatory and antiarthritic toothpaste was equally efficacious as three activity of S. lappa, A. speciosa and A. commercially popular toothpastes in terms aspera, Phytomed. 2012; 9(5): 433- of all evaluation properties of toothpaste. 437.

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