Tamarindus indica Fruit: Pharmacognostical Standardization, Detection of Contaminant, and In Vitro Antioxidant Activity.

OBJECTIVE: The main objective of the current study was to perform pharmacognostical, physiochemical analysis and in vitro antioxidant activity of Tamarindus indica Linn. (Family: Fabaceae) fruit.
MATERIALS AND METHODS: The present study deals with pharmacognostical characters as identification parameters of the fruit, which were subjected to macro- and microscopical studies. Physiochemical analysis was performed as per World Health Organization-recommended parameters. Toxicological parameters such as heavy metals, aflatoxins, and pesticide residue and presence of microbial count were analyzed. This study also deals with the thin layer chromatography/high performance thin layer chromatography fingerprinting and antioxidant activity.
RESULTS: The microscopy study revealed the presence of epidermis, vascular bundles, parenchyma cells, mucilage fiber, starch grain, and nonlignified fibers. Physiochemical parameters such as loss on drying, moisture content, ash value, and extractive value were also determined. Heavy metal, aflatoxin, total microbial load, and pesticide residues were found to be variable but under the permissible limits.
CONCLUSION: It is critical to analyze these parameters in each crude herbal drug before further processing to ensure their safety and efficacy for better approval at the international podium. This study revealed precise identification for the meticulous crude drug which will be valuable in detection and manage to adulterations of the raw material. Copyright:

Introduction

Drugs from natural sources have played a major part in prevention and alleviation of various pathologies afflicting mankind since the ancient era. These drugs are preferred these days due to lesser adverse effects related with their use; they have more structural variety that leads to the discovery of novel drug molecules and present authenticated methods for pharmacognostical study as recommended by World Health Organization (WHO). Pharmacognostic and phytochemical analysis is compulsory for understanding the therapeutic efficiency of natural source drugs and validating the efficacy and safety profile of the crude drug.[1234] The alternative and traditional drugs provide for the health requirements of about 85% of the world population. It is necessary to sustain the quality, safety, and efficacy of herbal drugs and their products to avoid any severe health problems.[5] Indian healthcare comprises of medical pluralism, and ayurveda still stays prevalent in comparison to modern drugs, especially for cure of an assortment of chronic sickness conditions.[6]

WHO describes traditional drugs as including different health practices, approaches, knowledge, and ideas incorporating herb-, animal-, and/or mineral-based medicines, religious remedy, manual procedures, and exercises applied singularly or in combination to sustain well-being as well as to cure, diagnose, or avoid sickness.

Tamarindus indica Linn., commonly known as Tamarind and belonging to the family Fabaceae, is a long-living, huge, evergreen or semi-evergreen tree, 25–35 meters high with a wide trunk up to 1.0–2.5 meters. The fruit contains succinct acid, tartaric acid, grape acid, apple acid, citric acid, and pectin as well as invert sugar. The ripe fruit has 50%–60% edible pulp and contains fiber, carbohydrate, iron, water, protein, fat, ash, calcium, phosphorus, thiamine, vitamin C, riboflavin, and niacin. Fruit pulps have invert sugar, vitexin, isovitexin, vitamin B3, pipecolic acid, citric acid, nicotinic acid, 1-malic acid, pipecolic acid, orientin, isoorientin, volatile oils, pectin, cinnamates, serine, beta-alanine, praline, phenylalanine, lipid, leucine, and potassium.[7] The fruits are eaten up raw or prepared into a refreshing drink; the fruit pulp is a chief ingredient of Middle East and Indian cuisine. Fresh and dried fruits are consumed as a sour-flavoring agent in curries, fish, and sauces. They are bitter and sweet in taste, purgative, and carminative. The leaves and flowers are used to treat cough, fever, constipation, stomach pain, indigestion, urinary tract infection, and flatulence. The fruit pulp is employed as a main component in cooking preparations such as chutneys, curries, and ice cream in states where the tree grows naturally.[8910] The fruit of T. indica displays antimicrobial,[11] antidiabetic,[12] anti-inflammatory,[13] and antiasthmatic activity.[14]

However, no systematic study has been carried out on the plant leaves. Considering the importance of this plant, the aim of the current study was to evaluate different pharmacognostical properties such as macroscopical, microscopical, and physiochemical characterization, TLC profiling and antioxidant activity of the fruit of the T. indica as a whole and its powdered form.

Materials and Methods

Plant material

The fruits of T. indica were collected from local markets of New Delhi, India and authenticated at the School of Science, Jamia Hamdard, New Delhi with a voucher specimen (TI/FP-368) that was deposited in the herbarium of Hamdard University New Delhi.

Reagent

All the reagents were analytical grade and obtained from SD Fine Chemicals Ltd., Mumbai, India. Cadmium, lead, arsenic, and mercury were purchased from Sigma (St. Louis, MO).

Macroscopical study

An organoleptic and external morphological character of freshly collected fruits was observed under magnifying lens and naked eyes.[15]

Microscopical study

Microscopical studies were performed on a thin hand section of fruit. Free hand cross transverse sections of fruit were taken and stained with safranine to confirm its lignifications. The same procedure follows for powder study. Coarse powder was used to evaluate microscopical character of fruit powder.

Physiochemical study

In this study, air-dried fruit powder was used for determination of physiochemical parameters such as loss on drying, moisture content by Karl Fischer titration, total ash value, water soluble ash value, acid insoluble ash value, and extractive value as per WHO guidelines.[16]

Preparation of extracts

Crude powder (20g) of drug was defatted separately with 300mL of petroleum ether by Soxhlet apparatus for 6h. The defatted powder drug (4 g) was then extracted separately with 100 mL of each solvent chloroform, methanol, water, and alcohol: aqueous (50:50) for 6 h by Soxhlet method and then filtered to attain respective extracts. These extracts were concentrated by a rotary evaporator (Buchi, R-215; Switzerland). 25mL of the extract was used to analyze the percent extractive values of fruit in special solvents. The remaining extract was kept in an airtight glass container at 5–10°C for further study.

Evaluation of contaminants

The heavy metal analysis for the presence of lead, cadmium, mercury, and arsenic was performed for the dried T. indica fruit powder. It was carried out by using atomic absorption spectrometer. The official methods of the Association of Analytical Communities were used for the determination of aflatoxins high performance thin layer chromatography (HPLC) and pesticides gas chromatography–mass spectrometry (GC-MS).[17] Microbial limit test was performed as per the standard method mentioned in WHO guidelines. It incorporated total fungal count, total microbial count, and presence of pathogens such as Staphylococcus aureus, Salmonella ebony, Escherichia coli, and Pseudomonas aeruginosa.[18]

HPTLC finger printing

The extracts (chloroform, methanol, aqueous: alcohol [1:1]) and aqueous were taken and concentrated on a rotary evaporator (Buchi, R-215; Switzerland) and then dried. A stock solution (5mg/mL) was readied in a different solvent, and properly diluted stock solution was spotted on precoated silica gel G60 F254 TLC plates (Merck, Germany) via CAMAG Linomat V (CAMAG) applicator; TLC plates were developed by solvent system toluene: ethyl acetate: formic acid (5:4:0.5; v/v/v) for chloroform, methanol, aqueous: alcohol (1:1) extracts and butanol: acetic acid: water (8:2:2; v/v/v) for aqueous extract. The plates were scanned by Scanner 3 (CAMAG) at 366nm. Every photograph of chromatograms was made with the help of Reprostar 3 (CAMAG) digital camera.[19]

Assessment of in vitro antioxidant activity by reversed-phase HPLC-DPPH method

The 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging effect was evaluated using the procedure given by Yen and Chen.[20] DPPH (100 µM) was suspended in double distilled water. Fresh stock solution was taken for experiment every day. The DPPH solution was used as control. The DPPH solution (1mL) was added to 1mL of fruit extract and standard (ascorbic acid) with 3mL of double distilled water. The solution mixture was shaken strongly and placed in dark light at room temperature for 10min. The decline in area of the resulting solution was observed at 517nm for 10min. The variation in the decrease of peak area between the control and the plant extract was applied for calculating the percent radical scavenging activity. Each result was triplicated.

The sample is filtered via 0.2 µm nylon membrane filter, and an aliquot (20 µl) of the sample is injected for HPLC study. The reversed-phase HPLC (Shimadzu HPLC) system consists of a pump (LC-10 Ai, Japan), a system controller (SCL-10AVP), and a diode array detector (SPD-M10 AVP). Data study and processing were done using class LC10 software (Version 1.6) with a total runtime of 15min. Evaluations were performed by a LiChrospher® 100 RP-18e column (250 mm × 4mm, 5 µM) (Merck, Darmstadt, Germany). Isocratic elution was performed by methanol/water (80:20, v/v) at a flow rate of 1mL/min.

where PAControl = peak area of control, PASample = peak area of sample.

Results and Discussion

In a few cases, plants that produce herbal drugs look similar to associated species. A thorough study of the macroscopical characters can be useful in distinguishing them. The macroscopy of a herbal medicine includes its visual appearance to the naked eyes as well as through a magnifying glass. It usually depends on the part of the crude plant from which the drug is found or extracted. The fruit of T. indica is elongated rod and curved in shape with brown color. It measures 12–15cm in length and has a hard and rough surface with a brittle texture. It has a characteristic odor but tastes sweet and sour [Figure 1].

Figure 1

Tamarindus indica fruit

Anatomical Characters of the Plant Fruit

The transverse section of T. indica fruit showed the presence of very clear and well-defined epidermis. Ground tissue showed the presence of vascular bundles arranged in a collateral manner although the alignment of xylem and phloem was not that much clear. Parenchyma cells were present throughout within the ground tissue interspersed with fibers [Figure 2].

Figure 2

Transverse section of fruit of Tamarindus indica

Powder microscopy

The characteristic features of powder herbal drug are principally used in the identification and detection of the drug sample in the form of powder. Powder microscopy of T. indica showed cork cells and loosely arranged parenchyma cells. A fragment of mesocarp showed vessels and mucilage fiber; brown matter with mucilage cells were also present. Simple starch grain; rounded starch grain of 7–10 microns in diameter; nonlignified fibers and stone cell as solitary or in groups were observed in powder study [Figure 3].

Figure 3

Micro photograph of fruit powder of Tamarindus indica. (A) Cork cell in surface view, (B) parenchyma cell, (C) fragment of mesocarp showing vessels and mucilage fiber, (D) brown matter with mucilage cells, (E) starch grain, (F) nonlignified fiber, and (G) stone cell

Physiochemical evaluation

Different physiochemical parameters were tested and are represented in Table 1 with standard deviation. The evaluation of water is important to check the product quality. The fundamental principle involves the Bunsen reaction between iodine and sulphur dioxide in an aqueous media. Karl Fischer revealed that this reaction may be changed to be used for the evaluation of water in a nonaqueous system that has an excess of sulpher dioxide. Total ash value may be used for the estimation of inorganic materials, such as oxalate, carbonates, phosphates, and silica. Heating causes the loss of organic components in the form of CO2 parting behind the inorganic materials. Ash value is an imperative characteristic of a crude drug, and this parameter helps detect the amount of adulteration as well as establish the quality and purity of the drug. There is an extensive variation in the ash values of various drugs but the difference mostly varies within slight limits in case of the same drug. The acid insoluble ash contains mostly of silica and high acid insoluble ash so representing the contamination with earthly components. The water soluble ash is used to analyze the quantity of inorganic elements. The quantity of an extract that a drug yields in a specific solvent is often an approximate measure of the quantity of particular constituents that the drug contains. The drug should be extracted with various solvents in order of their increasing polarity to get the correct and dependable values. Usually, petroleum ether, chloroform, methanol, water: alcohol (50:50), and water extractives are taken into consideration for fixing the standard of a drug. The petroleum ether extract contains fixed oil, resin, and volatile materials; however, once the extract is heated at 105°C until constant weight, the volatile oils evaporated leaving behind resin, coloring substances, and fixed oil. Alcohol may dissolve almost all the substances; it is generally applied for determining the extractive index for those drugs, which have alkaloids, glycosides, and resin. Water is used for the drug samples containing aqueous soluble materials as main constituents. The obtained results were found to be within limits and comparable with pharmacopoeial standards [21] [Table 1].

Table 1

Summary of physiochemical parameters of Tamarindus indica fruit (n = 5)

Parameters	% w/w (Mean ± Standard deviation)
LOD	30.54 ± 2.47 %
Moisture content	24.92 ± 1.28 %
Ash value
Total ash	7.95 ± 0.63 %
Acid insoluble ash	0.516 ± 0.18 %
Water soluble ash	5.25 ± 0.92 %
Successive extraction values
Petroleum ether	2.20 ± 0.35 %
Chloroform	1.62 ± 0.24 %
Methanol	25.82 ± 4.77 %
Water: alcohol (50:50)	28.02 ± 4.45 %
Water	30.22 ± 5.40 %

LOD = loss on drying

Detection of contaminant

Contamination of traditional medicines by heavy metals is of main concern because of the toxicity, persistence, and bioaccumulative nature of such metals.[22] It is well known for its adverse effects on many parts of the body. Progressive exposure to lead results in a decrease in the performance of the nervous system and affects renal clearance.[23] Contribution of therapeutic active plant materials along with heavy metals (lead, cadmium, arsenic, and mercury) may be attributed to several reasons like environmental contamination and traces of pesticide. The sample was found to pass the normal permitted limits of heavy metals [Table 2].

Table 2

Heavy metals analysis of Tamarindus indica fruit (n = 5)

Name of the metal	Mean ± Standard deviation (ppm)	Limit (safe up to) (ppm)
Lead	0.56703± 0.0085	10
Cadmium	0.00475 ± 0.0002	0.30
Mercury	0.431 ± 0.0281	0.50
Arsenic	1.180 ± 0.0171	3.0

Aflatoxins are mycotoxins formed by some fungus of the genus Aspergillus that may grow on numerous foods, spices, and therapeutic herbs. There are four main categories of aflatoxins—B1, B2, G1, and G2. The B1 and G1 aflatoxins are more toxic than aflatoxins B2 and G2 because of the presence of extra double bond that leads to the generation of an electrophilic reactive epoxide throughout liver metabolism. These may be the basis of acute structural and functional injury to the important organs of the human body. Aflatoxin B1 is considered a dangerous factor in the etiology of hepatocellular cancer in humans.[2425] Several ecological conditions, such as high moisture and temperature throughout the production and storage of medicinal plants, support the expansion of aspergillus genus and the consequent contamination with aflatoxins. These aflatoxins were analyzed using HPLC method in T. indica and observed for their presence in T. indica. The results showed that there was no aflatoxin present in T. indica.

The ever-increasing utilization of herbal drugs requires large-scale cultivation of medicinal plants, which is not possible without the application of pesticides. Attentions generally focused on contamination by organochlorine pesticides because of their toxicity and persistence in environment and contamination by general pesticides.[2627] The pesticide was analyzed by GC-MS technique. It was observed on screening that the samples of T. indica are free from the 43 standard pesticides. Medicinal plant material normally carries a good range of microorganism and moulds usually originating from the soil. For insuring safety of the drug, bio burden level was determined by the method prescribed by WHO. The microbial profile of T. indica was observed to be satisfactory with total microbial plate count, moulds and yeast counts being 40 colony forming units (CFU)/mL (under WHO limit of not more than (NMT) 10 CFU/mL), total mould and yeast were 11 CFU/mL (under WHO limit of NMT 10 CFU/mL). Moreover, the pathogenic bacteria Escherichia coli, Salmonella, Pseudomonas, and Staphylococcus were found to be absent [Figure 4].

Figure 4

Microbial load study in Tamarindus indica fruit

TLC and HPTLC are main techniques by which the quality control and fingerprint of plant drug can be sustained. TLC/HPTLC has exceptional resolution and, thus, allows instantaneous detection of an extensive series of materials in a single run. They also assist in determining the individual herbs in poly-herbal formulations. The chief purpose of the TLC/HPTLC evaluation of T. indica was to develop exceptional TLC spots in the formulation as identifier of its every ingredient. Different mobile systems were tried by hit and trial method for different extracts. Satisfactory separation of constituents was obtained in mobile system toluene: ethyl acetate: formic acid (5:4:0.5; v/v/v) and butanol: acetic acid: water (8:2:2; v/v/v) for chloroform extract, methanol extract, aqueous: alcohol extract (50:50) and aqueous extract, respectively. The samples were applied and chromatogram was developed in respective solvents. The chromatogram scanned at 366nm, and the result was found to be satisfactory [Figure 5 and Table 3].

Figure 5

HPTLC chromatograms of (A) chloroform, (B) methanol, (C) aqueous: alcohol (1:1), and (D) aqueous extracts of Tamarindus indica fruit

Table 3

HPTLC fingerprint data of different extracts of Tamarindus indica fruit

S. No.	Sample	Solvent system	No. of peaks and Rf values
1	Chloroform extract	Toluene: Ethyl acetate: formic acid (5:4:1; v/v/v)	(07); 0.12, 0.19, 0.31, 0.42, 0.52, 0.68, 0.87
2	Methanolic extract	Toluene: Ethyl acetate: formic acid (5:4:1; v/v/v)	(10); 0.04, 0.07, 0.13, 0.20, 0.33, 0.52, 0.54, 0.62, 0.71, 0.84
3	Aqueous : Alcohol extract (1:1)	Toluene: Ethyl acetate: formic acid (5:4:1; v/v/v)	(11); 0.05, 0.09, 0.15, 0.20, 0.28, 0.34, 0.41, 0.50, 0.69, 0.75, 0.89
4	Aqueous extract	Butanol: acetic acid: water (8:2:2; v/v/v)	(12); 0.05, 0.09, 0.16, 0.24, 0.28, 0.32, 0.40, 0.54, 0.71, 0.80, 0.86, 0.89

It is well known that the HPLC–DPPH method is used for a quick evaluation of pure active antioxidant constituent in complex mixtures, mainly herbal extracts. The quicker the absorbance reduces, the more potent antioxidant efficacy of the constituent will be in terms of hydrogen-donating ability.[28] This study shows that the extracts have the proton-donating capability and could provide as free radical inhibitors or scavengers, acting probably as main antioxidants. A well-resolved, balanced, and sharp peak was found for DPPH at 8.175 ± 0.873min retention time [Figure 6]. It was found that the aqueous extract of T. indica diluted at 1000 μg/mL had radical scavenging activity of 81.48 ± 3.13% which was comparable to that of the standard ascorbic acid (100 μg/mL) (95.11 ± 3.43%). This result confirms that the method can be applied for a rapid screening of antioxidant compounds or more accurately radical scavenging activity of phytoconstituents.

Figure 6

HPLC chromatograms of (A) DPPH as control, (B) DPPH with ascorbic acid, and (C) DPPH with Tamarindus indica fruit extract

Conclusion

The present study may be helpful to complement data with respect to the identification, authentication, and standardization of crude plant drugs. It is also valuable for the estimation of antioxidant activity in poly-herbal formulations. In other words, the pharmacognostical features evaluated in this study may provide as a tool for identification of the plant for validation of the raw material and for standardization of its formulations at herbal industrial level in the future.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.