Physicochemical and phytochemical standardization of berries of Myrtus communis Linn.

PURPOSE: Herbal medicines are gaining more and more attention all over the world due to their long historical clinical practice and less side effects. The major limitation with herbal medicines is that the lack of standardization technique. Initially, the crude drugs were identified by comparison only with the standard description available.
MATERIALS AND METHODS: Standardization of drugs means confirmation of its identity and determination of its quality and purity. The quality control standards of various medicinal plants, used in indigenous system of medicine, are significant nowadays in view of commercialization of formulations based on medicinal plants. The quality of herbal drugs is the sum of all factors, which contribute directly or indirectly to the safety, effectiveness, and acceptability of the product. Lack of quality control can affect the efficacy and safety of drugs that may lead to health problems in the consumers. Standardization of drugs is needed to overcome the problems of adulteration and is most developing field of research now. Therefore, there is an urgent need of standardized drugs having consistent quality.
RESULTS: The drug showed the presence of phyto-chemical constituents. Powdered drug was treated with different reagents and examined under UV light. Different reagents showed different colors of the drug at 2 wavelengths. The percentage of physiological active compounds viz. total phenolics, tannins, volatile oil, fixed oil, and alkaloids were also observed.
CONCLUSION: Myrtus communis L. (Family: Myrtaceae) is one of the important drug being used in Unani system of medicine for various therapeutic purposes. In this study, an attempt has been made to study berries of M. communis from physico-chemical and phytochemical standardization point of view.

It is now realized that herbal medicines are safe, free from adverse effects. According to WHO 2002 report,[1] 80% of world population is still dependent on herbal remedies to treat illness. In the present scenario, the demand for herbal products is growing exponentially throughout the world, and major pharmaceutical companies are currently conducting extensive research on plant materials for their potential medicinal value. Therefore, quality control for the efficacy and safety of herbal products is essential. Myrtus communis or common Myrtle (Myrtaceae) is an ever green shrub or small tree with evergreen leaves, 6-8 feet in height with small foliage and covered with a deep fissured bark. The Myrtle (Myrtus) is a genus of 1 or 2 species of flowering plants, native to Southern Europe and North Africa and widespread in the Mediterranean area. It is cultivated in North West India gardens for its fragrant flowers.[23] Myrtle has some medicinal properties e.g., as an antiseptic and disinfectant with its balsamic properties. The aromatic and medicinal qualities of M. communis contribute to its use in pharmaceutical, cosmetics, and food products. Berries of M. communis are carminative and given in diarrhea, dysentery, hemorrhage, internal ulcerations and in rheumatism.[2-7] Myrtle berries are also used to produce myrtle liqueur, and a wine characteristic of Mediterranean areas.[4] In India, separate department of AYUSH (Ayurvedic, Unani, Siddha, Homeopathy, under ministry of health and welfare, Government of India) has been made. The main purpose of this department is to deal with the rules and regulations for the herbals along with the Drugs and Cosmetic Act and Rules for the implementation of Good manufacturing practices (GMP) in herbals. The steps are taken by this department not only help to make the quality of herbal products but also to safeguard the adverse effects of the herbals too.[6-9] The present study includes the determination of physico-chemical constants, preliminary phytochemical studies, fluorescence analysis, quantitative analysis and high-performance thin-layer chromatography (HPTLC) fingerprints of the successive extractives of the powder of berries of M. communis L.

Materials and Methods

Plant material

The dried berries of M. communis were procured from the Khari Bawli, Delhi. The identity of the berries was established by NISCAIR (National Institute of Science Communication and Information Resources), Pusa Gate, New Delhi. The Voucher specimen (NISCAIR/RHMD/CONSULT/-2007-08/914/98, dated 13/11/2007) of the test drug has been retained and deposited for the future reference in the department of Ilmul Advia (Pharmacology), Faculty of Unani Medicine, Hamdard University, New Delhi.

Physico-chemical and phytochemical investigations

The drug was coarsely ground in an electric grinder and passed through 60 mesh sieve to get a fine powder, and was then subjected to standardization for an evaluation of purity and strength. The dried berries of M. communis were examined morphologically followed by other parameters. Extractive values were determined for cold, hot, and successive extraction methods. Calibrated digital pH meter was used to determine the pH of 1% and 10% of drug solution. Standard methods were adopted for total acid, water soluble and acid insoluble ash. The powder of the berries was subjected to the fluorescence analysis after being treated separately with different solutions and examined under UV light as well as ordinary light. Different colors were observed and noted. Phytochemical screening of the powdered berries was done to detect different plant constituents such as saponin, tannin, flavonoids, proteins, and alkaloids. All the parameters were determined by the methods described in recognized pharmacopoeias and researchers used.[9-18]

Test for saponin

(i) Foam test

A 50 mg of the test residue is taken in a test tube and shaken vigorously with a 20 mg of sodium bicarbonate and water. If characteristic honeycomb like froth is obtained, it indicates the presence of saponins.

(ii) Hemolysis test

A little of the test residue was dissolved in normal saline in such a way that 5 ml of the solution represented 1 gm of the crude drug. In a series of 5 test tubes, doses of 0.2 ml, 0.4 ml, 0.6 ml, 0.8 ml and 1 ml were added, and volume was made up to 1 ml in each case with normal saline. 1 ml of diluted blood (0.5 ml of rat's blood diluted to 25 ml with normal saline) was added to each tube and changes observed. If hemolysis of blood occurs, saponins are present.

Test for tannin

(i) Ferric chloride reagent

A 5% w/v solution of ferric chloride in 90% alcohol is prepared. Few drops of this solution is added to a little of the above filtrate. Dark green or deep blue color indicates the presence of tannins.

(ii) Lead acetate test

A 10% w/v solution of basic lead acetate, dissolved in distilled water, was added to the test filtrate. If precipitate is obtained, tannins are present.

(iii) Potassium dichromate test

If on addition of a solution of potassium dichromate in a test filtrate, dark color is developed, tannins are present.

Test for flavanoid

(i) Shinoda test

A small quantity to test residue is dissolved in 5 ml ethanol (95%v/v) and reacted with few drops of concentrated hydrochloric acid and 0.5 gm of magnesium metal. The pink, crimson or magenta color is developed within a minute or two if flavonoids are present.

Test for protein

(i) Biuret test

A few mg of the residue is taken in water, and 1 ml of 4% sodium hydroxide solution is added to it. A drop of 1% solution of copper sulfate is followed after this. Violet or pink color is formed if proteins are present.

(ii) Xanthoproteic test

A little residue is taken with 2 ml of water, and 0.5 ml of concentrated nitric acid is added to it. Yellow color is obtained if proteins are present.

Test for alkaloid

Mayer's Reagent (Potassium mercuri-iodide solution)

It gives a pale yellow precipitate with the test residue.

Wagner's Reagent (Iodine solution)

It gives a brown or reddish brown precipitate with the test residue.

Hager's Reagent (a saturated solution of picric acid in cold water)

It gives characteristic crystalline precipitate with the test residue.

Quantitative estimations of total phenol, tannins, alkaloids, essential oil, fixed oil, and glycoside were carried out as follows:

Total phenol: For the extraction of the phenolic component, the fat-free sample was boiled with 50 ml of ether for 15 min. 5 ml of the extract was pipette into a 50 ml flask, then 10 ml of distilled water was added. 2 ml of ammonium hydroxide solution and 5 ml of concentrated amyl alcohol were also added. The samples were made up to mark and left to react for 30 min for color development. The absorbance of the solution was read using a spectrophotometer at 505 nm wavelengths.[19]

Total tannins: 500 mg of the sample was weighed into 100 ml plastic bottle. 50 ml of distilled water was added and shaken for 1 h in a mechanical shaker. This was filtered into a 50 ml volumetric flask and made up to the mark. Then, 5 ml of the filtrate was pipette out into a tube and mixed with 3 ml of 0.1 M FeCl 3 in 0.1 N HCl and 0.008 M potassium ferrocyanide. The absorbance was measured in a spectrophotometer at 120 nm wavelength, within 10 min. A blank sample was prepared, and the color also developed and read at the same wavelength. A standard was prepared using tannin acid to get 100 ppm and was measured.[19]

Total alkaloids: 5 g of the sample were weighed into a 250 ml beaker and 200 ml of 20% acetic acid in ethanol was added and covered to stand for 4 h. This was filtered, and the extract was concentrated using a water bath to one-quarter of the original volume. Concentrated ammonium hydroxide was added drop-wise to the extract until the precipitation was complete. The whole solution was allowed to settle, and the precipitate was collected by filtration and weighed.[19]

Total essential oil: The essential oils were isolated from 100 g of powder by hydro distillation for 3 h, using a Clevenger-type apparatus, extracted with etnaol, and dried (anhydrous Na 2 SO4) according to the method of Prakash et al.[20]

Total fixed oil: The fixed oils were extracted from powder using the continuous Soxhlet extraction technique with petroleum ether for 3 h. The extracts were filtered and concentrated under reduced pressure at 40°C.[21]

For fluorescence analysis, powder of berries was examined under UV light according to the method described by Chase and Pratt.[22]

Results and Discussion

Morphologically, the fruit is a 2 or 3-celled black, fleshy berry, each cell containing 4 or 5 reniform, hard, kidney-shaped, whitish seeds. The M. communis berries are of varying sizes and shapes (orbicular or ovoid-ellipsoid) with persistent calyx forming a crown. The seeds show 3 distinct portions with seed coat, endosperm, and embryo. The seed coat is dull yellow in surface view. The morphological characteristics were in accordance with the previous findings.[35-7] Organoleptic characters, moisture content, extractive values, pH values, ash values, crude fiber content as well as swelling index of the drug were determined, and their observations were depicted in Table 1. The amounts of extracts obtained by exhausting crude drug are indicative of approximate measures of their chemical constituents. Water-soluble extractives are indicative of water-soluble active constituents of crude drugs, such as tannins, sugars, plant acids, mucilage, glycosides etc. Alcohol is an ideal solvent for extraction of various chemicals like tannins, resins etc. Ether-soluble extractives are indicative of volatile and non-volatile substances. The moisture content of a drug should be minimized in order to prevent decomposition of crude drugs, either due to chemical change or microbial contamination. The percentage of moisture content ranging from 10 - 20% shows an ideal range for minimum bacteria as well as for fungal growth. Ash value is a criterion to judge the identity or purity of crude drugs. The drug was screened qualitatively for phyto-chemical constituents i.e. alkaloids, flavonoids, saponins, tannins and proteins. Similar observations were also reported earlier.[1821] Results of the preliminary phytochemical screening are depicted in Table 2. Powdered drug was treated with different reagents and examined under UV light. Different reagents showed different colors of the drug at 2 wavelengths [Table 3]. The powdered drug was treated with different reagents, and the colors shown by that treatment were observed, and results were presented in Table 4. The percentage of physiological active compounds viz. total phenolics, tannins, volatile oil, fixed oil, and alkaloids were reported in Table 5. Results of the present study are in agreement with the observations earlier reported.[421] HPTLC (High Performance Thin Layer Chromatography) is an important parameter, which is helpful in the purity and identification of drugs. It is usually quicker and gives better separation. HPTLC is a valuable tool for reliable identification because it can provide chromatographic patterns and can be stored as electronic image. Various successive extractives of M. communis were subjected to HPTLC analysis. Silica gel 60 F254, TLC plates (10 × 10 cm, layer thickness, 2 mm, E-Merk, Germany) were used as stationary phase. A camag HPTLC system containing Linomat V sample applicator, Hamilton syringe (100 μl), Camag TLC scanner-3 with WINCAT software, Camag twin trough chamber (20×10 cm) were used for present study. The plates were scanned at 254 nm and 366 nm in absorbance mode using Camag HPTLC scanner.[23] The details of HPTLC profiles of the drug were mentioned in Table 6 and Figures 1–4.

Table 1

Physico-chemical constants of berries of M. communis

Table 2

Results of preliminary phytochemical tests for M. communis

Table 3

Fluorescence analysis of drug powder

Table 4

Powdered drug reaction with different reagents

Table 5

Quantitative analysis of M. communis

Table 6

HPTLC chromatogram of successive extracts of M. communis

Figure 1

HPTLC chromatograms of berries of M. communis: Petroleum ether extract (366 nm)

Figure 4

HPTLC chromatograms of berries of M. communis: Methanolic extract (254 nm).

HPTLC chromatograms of berries of M. communis: Petroleum ether extract (254 nm)

HPTLC chromatograms of berries of M. communis: Chloroform extract (254 nm)

Conclusion

Physico-chemical standards are of great significance in assuring the quality, authenticity as well as purity and thereby, efficacy of the drug. Standardization of drugs is very essential to provide good quality drugs of high efficacy and potency. Such kind of standardization studies are needed in promoting the safe use of genuine drugs, thus, contributing to human health. The methods used for standardization help in identification and collection of genuine drugs and discard of the exhausted, adulterated, and spurious drugs. Thus, it can be concluded that the parameters studied in the present work may be used for quality evaluation and standardization of crude drug to achieve genuine and standard drug for therapeutic purpose.