Phytochemical and Physical Powder Characteristic Evaluation of Poly Herbal Formulation

Ankit Yadav*, Neeraj kumar, Abhishek Kumar, Chetan, Ajay kumar Garg.

School of Pharmacy, Raffles University, Neemrana Behror - Kotputli, Rajasthan-301705.

*Correspondence: ajay.pharma3006@gmail.com

INTRODUCTION

India is known as the diabetes capital of the world. Diabetes mellitus (DM) is a metabolic condition that causes hyperglycemia, insulin resistance, and abnormalities in carbohydrate, lipid, and protein metabolism. Its prevalence is rapidly increasing over the world, and it is likely to lead to serious secondary problems such as neuropathy, nephropathy, retinopathy, cardiovascular disease, retinopathy, and dyslipidemia in the long run. In today's world, around 90% of the young population accounts for a significant portion of the occurrence of type II diabetes, owing to a change to a sedentary lifestyle consisting of unhealthy eating habits and decreased physical exercise. Various synthetic medications, including oral hypoglycemic drugs and insulin, are available to manage blood sugar levels, but their high cost, problems, poor tolerance, and a variety of adverse effects prevent widespread adoption. Thus, it is particularly one of the resistant diseases defined by the Indian Council of Medical Research, and there is a great need for alternative medical treatments [1], [2], [3]. Given the facts, phytotherapy is the most financially successful and commonly used field of alternative or supplementary medicine, resulting in a synergy that is more powerful than its parts. India is known as a medical plant emporium because, in various bioclimatic zones, thousands of medicinal plants are available, and the country has a long history of employing herbal plants for medicinal purposes. Traditionally, herbal medications and their preparations have been employed in many therapies because of their natural origin and lower adverse effects than synthetic drugs. [4], [5], [6] Phytotherapy thrives when more than one herb is used in the formulation, resulting in increased therapeutic effectiveness known as polyherbalism. To obtain the synergistic effect, either pharmacodynamic or pharmacokinetic synergism is necessary, which means that either the herb will target the therapeutic activity to a receptor or will facilitate the absorption, distribution, metabolism, and elimination of the other herbs. This study examined the pharmacological and physiological properties of a Polyherbal powder (PHP). Some PHP plants with the potential to treat DM are discussed. Ocimum sanctum or O. tenuiflorum (family: Lamiaceae), often known as holy basil or tulsi, exhibits pharmacological actions such as antioxidant, anti-diabetic, anti-inflammatory, antihypertensive, cardio-protective, hepato-protective, and reno-protective. It is widely regarded and one of the most powerful traditional plants for diabetes treatment. Tulsi leaf extract has been demonstrated to stimulate insulin secretion from isolated islets, perfused pancreas, and colonal pancreatic β-cells, indicating an anti-hyperglycemic action. [3], [7], [8]. Amla, also known as Emblica officinalis or Phyllanthus emblica (Euphorbiaceae), is a well-studied plant rich in tannins, alkaloids, and phenols. Its fruit juice contains the highest concentration of vitamin C (ascorbic acid). The high content of vitamin C helps to regulate diabetes. It is said to be helpful at lowering fasting blood glucose, postprandial blood glucose, and HbA1c levels. Scientists have discovered a proposed mechanism for how it operates as an antidiabetic and reduces problems. Ellagic acid effectively inhibits α-amylase and α-glycosidase enzymes. It has been shown to increase biomarkers of oxidative stress (nitric oxide, glutathione, and malondialdehyde), HbA1c levels, and high sensitivity. C-reactive protein levels[9], [10]. Cinnamomum zeylanicum (Lauraceae), often known as dalchini, has been demonstrated to boost insulin levels in glucose metabolism. It has been shown to improve glucose absorption by activating insulin receptor kinase, autophosphorylation of the insulin receptor, and glycogen synthase activity. [11]

Turmeric (Curcuma longa L.) is a plant from the Zingiberaceae family that is used to treat inflammation. It's a relatively tall perennial plant with underground rhizomes. Rhizomes are often oval, pyriform, oblong, and frequently short-branched. It is commonly used as a flavoring, preservative, and coloring agent in South Asia, India, and China. It is well-known for having unusual therapeutic characteristics. It is grown in tropical areas like as Pakistan, China, Peru polyphenols, and India. Curcuma longa is considered a native of India. It is commercially farmed in various south Asian countries, including China and India. It is well-known for its culinary use as a fundamental component of curry powder. It is an authorized food ingredient in the United States. Turmeric is commonly known as haldi or haridra in India. It is also known as "Manjal" and its powder as Manjal Thool in Tamil. It is also known as "Indian saffron" since Curcuminoids are widely used as a substitute for the more expensive saffron spice. [12]

Materials and methods

Collection & preparation of plant material:

The Raffles University, Neemrana provided O.Sanctum, E. officinalis leaves, C. Zeylanicum barks, and C. longa rhizomes were purchased fresh from the local market. These raw materials were shade dried and thoroughly ground in an electrical grinder. They were blended in a precise ratio with a maximum of O. sanctum, C. zeylanicum, and the remaining herbs were added at half the amount of the above-mentioned herbs. Organoleptic, Physicochemical and phytochemical evaluation [13], [14]. Organoleptic evaluation is the evaluation of a product based on its color, odor, taste, and texture. The organoleptic examination followed the Wallis approach. The physicochemical characteristics such as moisture content, pH, and ash value were determined. PHP was also submitted to preliminary phytochemical screening to detect the presence of organic elements utilizing established procedures.

Figure 1: a) Amla fruits and leaves                           b) E. officinalis (Leaves)

Figure 2: a) Tulsi (Oscimum Sanctum)

Figure 3: Curcuma Longa

Figure 4   Cinnanomum Zeylanicum

Moisture Content:

Drying loss is used to control moisture content, as excess moisture can lead to hydrolytic reactions and microbiological growth. The moisture content was determined using the gravimetric method, and the loss during drying was computed. Two grams of PHP were placed in a weighted warmed porcelain dish, and then dried at 105° in a hot air oven until a consistent weight or two consecutive weights varied by 0.5 mg were recorded. After drying, the weight was taken and transported to the desiccators to cool before the porcelain dish was weighed again. To compute percent moisture content, use the equation (% moisture content = (W1-W2)/W) × 100, where W is the sample weight (2 g), W1 is the weight before drying, and W2 is the weight after drying.

Ash content:

Ash readings typically reflect inorganic residues such phosphates, carbonates, and silicates found in herbal medications. These metrics indicate the quality and purity of herbal medicines. The goal of evaluation is to remove any organic materials that may interfere with analytical results.

Total ash:

The empty silica crucible was weighed (W1). The previously weighed crucible received approximately 3 g (W2) of air-dried PHP. To detect the absence of carbon, the sample was gradually ignited in an electrical muffle furnace and heated to 500-600° until it turned white. Then it was chilled in a desiccator and weighed again. The formula for calculating total ash content is ((W3-W1)/(W2-W1))×100.

Acid-insoluble ash:

Dilute HCL (25 milliliters) was applied to the crucible containing complete ash. The mixture was slowly boiled for 5 minutes while covered with a watch glass. Wash the watch glass with 5 ml of hot water, and then add it to the crucible. To obtain a neutral filtrate, insoluble materials were filtered using ash less filter paper and rinsed with hot water. The filter paper with insoluble particles was transferred to the original crucible, dried on a hotplate, and burned to a constant weight (W4). After cooling for 30 minutes in desiccators, the residue was weighed again. W1 is the weight of an empty silica crucible, W2 is the weight of the sample with the crucible for ignition, and W3 is the W3 represents the sample's final weight, including the weight of the crucible after fire, while W4 represents the constant weight after adding HCL.

Acid-insoluble ash content was calculated as, % acid-insoluble ash = (W4–W1)/(W2–W1)×100.

Water-soluble ash:

To remove ash from the crucible, add 25 ml of water and boil for 5 minutes before filtering through ash less filter paper. The insoluble materials on the filter paper were rinsed with hot water before being fired in a crucible for 15 minutes at a temperature not exceeding 500°. After cooling in desiccators for 30 minutes, the residue was weighed again (W5). The percentage of water-soluble ash was calculated using the formula (W7-W6) × 100, where W1 is the weight of the empty silica crucible, W2 is the weight of the sample before ignition, W3 is the weight of the sample after ignition, W6 is the residue weight (W5-W1), and W7 is the weight of ash (W3-W1).

The water-soluble ash is W7-W6 mg/g.

Flow characteristics of powder (rheological parameters):

A pre-formulation study is a strategy used in drug development to gather information on the compound's qualities and propose a development timetable. The formed powder's rheological parameters were studied and estimated, including angle of repose, bulk density, tapped density, and compressibility index. The angle of repose was measured using the fixed funnel method. A funnel was put above graph paper on a flat horizontal surface and secured with its tip at a specific height (h). PHP was poured through the funnel until it touched the top of the conical mound. The conical pile's heap developed a radius (r) on its base. The angle of repose (θ) is calculated as θ= tan^-1 h/r,

Where, h is the cone's height, r is the radius of the base, and tan θ = h/r.

TABLE 1: flow characteristics of the powder

The angle of repose, Carr's index, and Hausner's ratio values determine the powder's flowability within a certain range.

Carr's Index measures the degree of powder compression, while Hausner's Ratio assesses the ease of powder flow. Determining true and tapped density is necessary for their calculation. Carr's index is determined using the following formula:

(ρtap-ρb)/ρtap)/×100.

 Hausner's ratio can be determined using the formula ρtap/ρb.

To determine bulk density, put 5 g of PHP (M) to a dry 100 ml cylinder, level the powder, and read the apparent volume (V0) without compacting. The bulk density (ρb) was estimated using the equation

ρb=M/V0.

Where, M is the sample's weight and V is the apparent volume of the powder.

To determine tapped density, tap the PHP 500 times, then 750 times, and 1250 times until the difference between measurements is less than 2%. The tapped volume (Vf) is then measured. The tapped density (ρtap) was computed in g/ml using the formula

ρtap = M/Vf,

 Where, M is the sample weight and Vf is the tapped volume of powder. Summary of powder flowability by angle of repose.

Phytochemical screening, test for alkaloids:

Phytochemical assays were conducted on PHP using established protocols to detect components.
Dragendorff's test was used to identify alkaloids. To 0.5 mL of aqueous PHP solution, add Dragendorff's reagent (potassium bismuth iodide solution). Reddish-brown precipitate indicates the presence of alkaloids. To perform the Hager's test, add a few drops of the reagent to 0.5 ml of aqueous PHP. A yellow precipitate indicates the presence of alkaloids. Adding Wagner's reagent (iodine in potassium iodide) to 0.5 ml of aqueous PHP solution resulted in a reddish-brown precipitate, indicating the presence of alkaloids. To conduct Mayer's test, 0.5 ml of aqueous PHP solution was mixed with Mayer's reagent.

Tests for carbohydrates:

To execute the molisch test, 0.5 ml of aqueous PHP solution was mixed with a few drops of alcoholic α-naphthol solution, then 0.2 ml of concentrated sulphuric acid was added along the edges of test tubes. The appearance of a reddish-violet ring at the confluence of the two layers suggested the existence of carbs. To reduce sugars. Benedict's test was completed by taking
the 0.5 ml aqueous PHP solution was shaken with 2.5 ml of water, filtered, and heated to concentrate. To the concentrated filtrate, add 5 mL of Benedict's solution and heat for 5 minutes. The formation of a brick-red precipitate indicates the existence of free reducing sugar. Fehling's test involved mixing equal quantities of Fehling's A (copper sulphate). Using Fehling's-B (potassium tartrate and sodium hydroxide in distilled water) and copper sulphate reagents, add a few drops of aqueous PHP solution and boil the mixture. A brick-red precipitate of cuprous oxide revealed the existence of free reducing sug.Monosaccharides was identified using the Barfoed test. Dilute 0.5 ml of aqueous PHP solution with distilled water, filter, and combine 1 ml of the filtrate with 1 ml of Barfoed reagent. Heat on a water bath for 2 minutes. The brick-red precipitate of cuprous oxide indicated the presence of monosaccharide’s. Starch was detected by a dark blue color when 0.5 ml of aqueous PHP solution was mixed with iodine reagent. The color faded on heating and reappeared on cooling.

Tests for flavonoids and glycosides:

To identify flavonoids, 5 ml of dilute ammonia was added to 1 ml of an aqueous PHP solution, followed by concentrated sulfuric acid. Flavonoids were identified by their yellow hue. Anthraquinone glycosides were identified using the Born Trager test. To 0.5 ml of aqueous PHP solution, add 0.5 ml of dilute ammonia and 1 ml of benzene. Anthraquinone glycosides were identified by their reddish-pink color. Keller Killiani's technique was used to detect cardiac glycosides. To prepare the aqueous PHP solution, 0.5 ml of concentrated sulphuric acid and 0.4 ml of glacial acetic acid with trace ferric chloride were carefully added. A reddish-brown color appears at the confluence between two layers.

Test for saponins, steroids and triterpenoids:

A pinch of dried PHP was added to 3 ml of distilled water and shaken briskly.The formation of foam suggested the presence of saponin. A positive Liebermann-Burchard test indicates the presence of steroids and terpenoids. To 0.5 ml of aqueous PHP solution, add a few drops of acetic anhydride, boil, and then cool. Concentrated sulphuric acid was applied to the walls of the test tube. Steroids were identified by a brown ring at the junction of two layers, whereas triterpenoids were identified by a deep red color. The upper layer was green. The Salkowski test involved adding chloroform to 0.5 cc of aqueous PHP solution and a few drops of strong sulfuric acid. It was shook and left to stand. The red color in the lower layer indicates the presence of steroids and their production.

Tests for tannins:

Tannins were discovered using the lead acetate assay. A few drops of 10% lead acetate were added to 0.5 ml of aqueous PHP solution. The formation of a precipitate indicates the presence of tannins. To test for tannins, add a few drops of 0.1% ferric chloride solution to 0.5 ml of aqueous PHP solution. Tannins are indicated by blue-black or brownish-green coloration.

Tests for phenolic compounds:

Adding a few drops of 10% lead acetate solution to the aqueous PHP solution resulted in the production of a white precipitate, indicating phenolic chemicals. A few drops of neutral 5% ferric chloride solution were added to 0.5 ml of aqueous PHP solution. The presence of phenolic chemicals resulted in a dark green color.

Tests for amino acids:

To do Millon's test, add 2 ml of Millon's reagent (mercuric nitrate in nitric acid with trace amounts of nitrous acid) to 0.5 ml of aqueous PHP solution. Gently heating caused a white precipitate to turn crimson, indicating the presence of amino acids. The ninhydrin test involved adding a few drops of 5% ninhydrin to 0.5 ml of aqueous PHP solution and heating it. The violet color indicated the presence of amino acids. Proteins were identified using the Biuret test, which involved adding 0.5 mL of aqueous PHP solution, 4% sodium hydroxide solution, and a few drops of 1% copper sulphate solution. Protein presence was shown by the appearance of violet hue. To identify oils and fats, press a tiny amount of PHP between two filter sheets. The oil stain on the filter sheets showed the presence of oils. To test for coumarins, 10% sodium hydroxide was added to 0.5 mL of aqueous PHP solution. Yellow color indicates the presence of coumarins.

RESULT AND DISCUSSION

The organoleptic characteristics of PHP were examined and shown in Table 2. Previous investigations on polyherbal formulations for DM found that a bitter taste affected patient acceptability [2], [5], [15], [16]. The PHP created in this study improves patient acceptability by combining particular herbs for a pleasant taste. Moisture is the primary cause of degradation in medications and formulations. Excessive moisture in plant medications can lead to bacterial and fungal growth, as well as metabolic reactions. The formulation with lower moisture content is expected to be more stable over time. Plant drugs require a moisture content of less than 14%. In this analysis, individual medicines and PHP had moisture content below 10%, ranging from 5±0.01-8±0.01% w/w (See Fig.1). The ash value is crucial for ensuring herbal medication quality. A high ash value may imply adulteration, contamination, replacement, or negligence in drug preparation. The investigation found little contamination, with total ash values ranging from 6±0.01 to 9±0.01% w/w. Water-soluble ash is a portion of the total ash content that dissolves in water. It can indicate faulty preparation or the existence of previously removed water-soluble salts in a medication. This refers to the weight differential between total ash.

TABLE 2: ORGANOLEPTIC PROPERTIES OF POLYHERBAL POWDER

Evaluation of PHP powder's organoleptic qualities, including colour, aroma, taste, and appearance. The water soluble ash values for individual drugs and PHP ranged from 2±0.01-3±0.01% w/w. The range of water-soluble ash showed normal medication quality.  Individual medicines and PHP showed acid-insoluble ash values ranging from 1±0.01-2.5±0.01% w/w. Figure 2 depicts the total ash value, water-soluble ash, and acid-insoluble ash for each particular medication and PHP.The pH of the aqueous solution of PHP was 6±0.02. Bulk density and tapped density analysis are crucial for powder packaging decisions.  Powder consolidation can be determined by tapping its density. Consolidation of powder leads to increased flow resistance. Our analysis indicated that tapping density ranged from 0.65±0.01-0.92±0.01% w/w. Thus, having a low tapping density. Figure 3 depicts the tapped density, bulk density, and Hausner's ratio for specific medications in the PHP. Carr's index and angle of repose reflect the powder's compressibility and free flow. Figures 4A and B depict the flow property of the powder.

Fig. 5: Moisture content of individual herbs and PHP Moisture content is expressed as % w/w and PHP is polyherbal powder.

Fig. 6: Percent ash values of individual herbs and PHP Percent ash values (%w/w) of individual herbs and the polyherbal powder (PHP).

Fig. 7: Bulk density, tapped density and Hausner’s ratio (%w/w) of individual herbs and PHP.

Fig. 8 -Angle of repose (% w/w)

Fig. 9: Carr’s Index  of individual herbs and the poly herbal powder (PHP).

Research suggests that C. zeylanicum can block α-glucosidase and α-amylase, leading to antihyperglycemic effects similar to those of O. sanctum. The high polyphenol and flavonoid content causes maltase inhibition and delayed glucose release in the bloodstream. Phytochemical research indicates that the aqueous extract of tulsi leaves contains cardiac glycosides, flavonoids, glycosides, and tannins. This study found that the phytochemical examination of PHP revealed the presence of active elements such glycosides, flavonoids, and tannins, which help reduce blood glucose levels. Active components, such as glycosides [11]. Research suggests that several herbs can reduce blood glucose levels through multiple processes. Seeds can lower blood glucose levels and avoid problems through several methods, as demonstrated by this PHP. T. foenum-graecum seeds can help treat diabetes through their alkaloid content, which prevents catabolic processes like glycogenolysis and lipolysis, modulates insulin secretion, and contributes to antioxidant activity. This can work in tandem with other herbs to lower blood glucose levels. We mostly use these herbs in our PHP composition. Table 3 shows phytochemical screening results for both PHP extracts.

TABLE 3: PHYTOCHEMICAL SCREENING OF THE POLYHERBAL POWDER

Evaluation of phytochemical components in an aqueous solution of polyherbal powder (PHP): (+) indicates presence, (-) indicates absence.

Flavonoids and tannins help reduce blood glucose levels. Research suggests that several herbs can reduce blood glucose levels through multiple processes. Seeds can lower blood glucose levels and avoid problems through several methods, as demonstrated by this PHP. C. Longa rhizomes can help treat diabetes through their alkaloid content, which prevents catabolic processes like glycogenolysis and lipolysis, modulates insulin secretion, and contributes to antioxidant activity. This can work in tandem with other herbs to lower blood glucose levels. We mostly use these herbs in our PHP composition. Phytochemical screening for both extracts. (Moisture content, flow properties, weight loss during drying, total ash, water-soluble and acid-insoluble ash). Standardization research findings can be used to assess formulation quality and purity. This formulation may be effective for treating diabetes.

ACKNOWLEDGEMENT:

We gratefully acknowledge our Professor, Dr. Gadangi Indira, for providing work facilities. We appreciate the cooperation of the institute’s Mentors, Principal and Other faculty members.

REFERENCES

[1]      R. Unnikrishnan, R. M. Anjana, and V. Mohan, “Diabetes mellitus and its complications in India,” Nat. Rev. Endocrinol., vol. 12, no. 6, pp. 357–370, 2016, doi: 10.1038/nrendo.2016.53.

[2]      T. Nagja, K. Vimal, and A. Sanjeev, “Anti-diabetic activity of a polyherbal formulation in streptozotocin induced type 2 diabetic rats,” J. Nat. Remedies, vol. 16, no. 4, pp. 148–152, 2016, doi: 10.18311/jnr/2016/15323.

[3]      R. A. Antora and R. M. Salleh, “Antihyperglycemic effect of Ocimum plants: A short review,” Asian Pac. J. Trop. Biomed., vol. 7, no. 8, pp. 755–759, 2017, doi: 10.1016/j.apjtb.2017.07.010.

[4]      P. PrabhuT and C. AtleeW, “Anti-inflammatory, anti arthritis and analgesic effect of ethanolic extract of whole plant of Merremia Emarginata Burm.F,” Cent. Eur. J. Exp. Biol., vol. 1, no. 3, pp. 94–99, 2012, [Online]. Available: http://scholarsresearchlibrary.com/archive.html

[5]      Thillaivanan S, “Challenges, Constraints and Opportunities in Herbal Medicines-A Review,” Int. J. Herb. Med., vol. 2, no. 1, pp. 21–24, 2014, [Online]. Available: http://scholarsresearchlibrary.com/archive.html

[6]      C. C. Falzon and A. Balabanova, “Phytotherapy: An Introduction to Herbal Medicine,” Prim. Care - Clin. Off. Pract., vol. 44, no. 2, pp. 217–227, 2017, doi: 10.1016/j.pop.2017.02.001.

[7]      P. Bhattacharyya and A. Bishayee, “Ocimum sanctum Linn. (Tulsi): An ethnomedicinal plant for the prevention and treatment of cancer,” Anticancer. Drugs, vol. 24, no. 7, pp. 659–666, 2013, doi: 10.1097/CAD.0b013e328361aca1.

[8]      L. I. G. Paula-Freire, G. R. Molska, M. L. Andersen, and E. L. D. A. Carlini, “Ocimum gratissimum Essential Oil and Its Isolated Compounds (Eugenol and Myrcene) Reduce Neuropathic Pain in Mice,” Planta Med., vol. 82, no. 3, pp. 211–216, 2015, doi: 10.1055/s-0035-1558165.

[9]      S. Dasaroju and K. M. Gottumukkala, “Review Article Current Trends in the Research of,” Int.J.Phara.Sci.Rev.Res, vol. 24, no. 2, pp. 150–159, 2014.

[10]    A. Ansari et al., “Emblica officinalis improves glycemic status and oxidative stress in STZ induced type 2 diabetic model rats,” Asian Pac. J. Trop. Med., vol. 7, no. 1, pp. 21–25, 2014, doi: 10.1016/S1995-7645(13)60185-6.

[11]    Gaber E. El-Desoky, “Antidiabetic and hypolipidemic effects of Ceylon cinnamon (Cinnamomum verum) in alloxan-diabetic rats,” J. Med. Plants Res., vol. 6, no. 9, pp. 1685–1691, 2012, doi: 10.5897/jmpr11.1472.

[12]    T. Nisar, M. Iqbal, A. Raza, M. Safdar, F. Iftikhar, and M. Waheed, “Turmeric: A Promising Spice for Phytochemical and Antimicrobial Activities,” J. Agric. Environ. Sci, vol. 15, no. 7, pp. 1278–1288, 2015, doi: 10.5829/idosi.aejaes.2015.15.7.9528.

[13]    V. B. Gawali, M. Bhalsingh, N. B. Dalvi, and Y. S. Tarkasband, “Development and evaluation of polyhebral powder formulation as energy booster,” J. Pharmacogn. Phytochem., vol. 7, no. 3, pp. 1576–1580, 2018.

[14]    A. I. Hussain, F. Anwar, P. S. Nigam, M. Ashraf, and A. H. Gilani, “Seasonal variation in content, chemical composition and antimicrobial and cytotoxic activities of essential oils from four mentha species,” J. Sci. Food Agric., vol. 90, no. 11, pp. 1827–1836, 2010, doi: 10.1002/jsfa.4021.

[15]    H. S. Chandel, A. K. Pathak, and M. Tailang, “Standardization of some herbal antidiabetic drugs in polyherbal formulation,” Pharmacognosy Res., vol. 3, no. 1, pp. 49–56, 2011, doi: 10.4103/0974-8490.79116.

[16]    M. S. Baliga et al., “Ocimum Sanctum L (Holy Basil or Tulsi) and its phytochemicals in the prevention and treatment of cancer,” Nutr. Cancer, vol. 65, no. SUPPL.1, pp. 26–35, 2013, doi: 10.1080/01635581.2013.785010.