Pharmacognostic study and development of quality control parameters for fruit, bark and leaf of Zanthoxylum armatum (Rutaceae).

CONTEXT: Zanthoxylum armatum (Rutaceae) fruit, bark and leaves are used for various conditions of ailments in traditional systems of medicine since ancient times. AIMS: This study is designed to lay down the various pharmacognostic and phytochemical standards which will be helpful to ensure the purity, safety, and efficacy of this medicinal plant.
MATERIALS AND METHODS: Various methods including macroscopic, microscopic, physicochemical, and phytochemical methods were applied to determine the diagnostic features for the identification and standardization of intact and powdered drug of Z. armatum leaf, fruit, and bark.
RESULTS: The shape, size, color, odor, surface characteristics were determined for the intact drug and powdered materials of leaf, bark and fruit of Z. armatum. Light and electron microscope images of cross-section of leaf and powdered microscopy revealed useful diagnostic features. Histochemical, phytochemical, physicochemical including fluorescence analysis of powdered drug proved useful to differentiate the powdered drug material. High performance liquid chromatography analysis showed the presence of important phytoconstituents such as gallic acid and rutin.
CONCLUSION: The data generated from this study would be of help in the authentication of various parts of Z. armatum, an important constituent of various herbal drug formulations. The qualitative and quantitative microscopic features would prove useful for laying down pharmacopoeial standards. Morphology as well as various pharmacognostic aspects of different parts of the plant were studied and have been described here along with phytochemical, physicochemical studies, which will help in authentication and quality control.

INTRODUCTION

According to WHO chapter of standardization and quality control of herbal drugs, standardization involves the physicochemical evaluation of crude drug covering aspects such as selection and handling of crude material. Most of the attention is normally paid to quality indices including macro and microscopic examination, ash values, moisture content, extractive values, crude fiber, qualitative and quantitative chemical evaluation and chromatographic examination.[12]

Wild plants form a major source of raw drugs for local communities and herbal drug industry. The raw materials are often adulterated when purchased from markets.[3]

Across the globe, herbal industries and local communities generally face the problems of adulteration and substitution at the raw material stage.[4] In this study, the herbal drug Zanthoxylum armatum (leaves, fruit and bark) was selected as a case study for methods to authenticate it based on taxonomic and pharmacognostic analyses.

Zanthoxylum armatum (Roxb.) belongs to family Rutaceae and is a very common plant of Southeast Asia.[5] It grows in Dir, Hazara, and Muree hills of Pakistan.[6] The aerial parts are of this plant are used as stomachic and carminative.[78] They are also used as a remedy for toothache.[7] Z. armatum branchlets are used as miswak/Tejbal (tooth brush) for cleaning of teeth, while its fruit powder is applied in toothache.[9] The seeds and the bark of this plant are used in the treatment of fevers, indigestion/heartburn, cholera and as a tonic.[7] Various species of Zanthoxylum genus possess antimicrobial, larvicidal and cytotoxic activity.[101112131415]

It is also reported that this plant posses abortifacient, antifertility,[16] antiseptic, disinfectant, deodorant[17] antipyretic, and anti-diarrheal activities. It improves speech in children and increases saliva secretion.[18] Fruits and seeds of this plant are used in fever, dyspepsia and skin diseases.[19]

The different classes of chemical constituents reported from Z. armatum include terpenes, sterols, flavonoids, alkaloids and coumarins.[62021222324]

MATERIALS AND METHODS

Plant material

Zanthoxylum armatum leaves, fruits and bark were collected from Tanawal area of district Haripur, Khyber Pakhtunkhwa province, Pakistan, in the month of August. It was authenticated by Prof. Dr. Manzoor Ahmed, Department of Botany, Postgraduate College, Abbottabad. Fresh leaves were used for microscopic study. The collected parts of the plant were washed with water and dried in shade at room temperature. The dried parts were ground to a coarse powder. The powdered drug was stored in an air tight and light resistant container for the study.

Macroscopic studies

Macroscopic and organoleptic studies were conducted on intact and powdered materials. All samples were washed, air dried in shade and observed for color, shape, odor, taste, size, fracture and other surface characteristics. Leaves which were shade dried for 10–15 days, fruit and bark were powdered and observed for color, odor, taste and for filter paper test.[25]

Microscopic studies

Morphological examinations were conducted using a binocular zoom light microscope, semi plan achro (Model AxL, LABO, Germany). Cross-sections were prepared by free hand sectioning cleared with chloralhydrate, stained with freshly prepared dyes safranin and fast green, and different grades of alcohol were used to increase visibility.[26] All the images presented were taken by the author using a Samsung digital camera (PL 211).

For quantitative microscopy (stomatal index, vein islet, vein termination number and palisade ratio), sections (5 mm × 5 mm) were cut, cleared with chloralhydrate and observed under a microscope.[26]

For powdered drug, shade dried material was ground and passed through sieve 10. The samples were observed in chloral hydrate and permanent slides were prepared using Canada balsam as mentioned by Trease and Evans.[25] For scanning electron microscopic (SEM) analyses, samples were attached to aluminum stubs and coated with gold (30–40 nm) and dried with CO2. The samples were examined under JEOL, JSM-5910, SEM (SEM interface version 5.05) at 20 kV.[27]

Physicochemical analysis

Total ash and acid insoluble ash contents are important indices to determine quality and purity of herbal medicines. Total ash values of leaves, fruit and bark, were determined by igniting the powdered sample gradually between 500°C and 600°C until it turned white, and the resultant powder was desiccated and weighed. Acid insoluble ash content was measured by solubilizing the part of total ash in hydrochloric acid with boiling followed by collecting and washing on filter paper, cooling in desiccator and weighing. Similarly, water soluble ash content was also measured. For extractive values, 2 g of each finely powdered sample were macerated with solvents of different polarities and was stirred on a shaker for 6 h. The extracts were filtered, concentrated to dryness and weighed. Ash and extractive values were determined as given in the guidelines of WHO.[26] The fluorescence analysis of powdered samples was carried out using ultraviolet (UV) lamps of short (254 nm) and long (365 nm) wavelengths. The samples were observed for fluorescence when mixed with different solvents and reagents. The fluorescence analysis was carried out by the method of Chase and Pratt.[28]

Loss on drying (LOD) values were measured by drying the air dried material (2–5 g) in the oven at 100–105°C. Drying was continued until the two consecutive weight measurements did not differ by 5 mg.[26]

Preliminary phytochemical screening

For phytochemical screening of alkaloids freshly prepared Dragendroff's and Mayer's reagents were used and observed for the presence of turbidity or precipitation as mentioned by Brain and Turner.[29] Test conducted to detect the presence of flavonoids were: Ammonia sulfuric acid, aluminum chloride, dilute ammonia, and potassium hydroxide tests as mentioned by Harborne.[30] For screening of tannins three tests, namely, gelatin, vanillin and ferric chloride tests were performed as mentioned by Ali.[31] To detect saponins, froth, emulsion and lead acetate tests were applied.[32] To detect the presence of steroids, acetic anhydride was used with sulfuric acid in the first test and acetic chloride with sulfuric acid were used in another.[30] Sample extracted with chloroform was treated with sulfuric acid to test for the presence of terpenoids.[30] Ammonia solution and ferric chloride solutions were used for anthraquionones testing as mentioned by Brain and Turner.[29]

Histochemical tests for detection of cellulose cell walls, lignified cell walls, cuticular cell walls, aleurone grains, calcium carbonate, fats, oils and resins, hydroxyl quinines, inulin, mucilage, starch and tannins were carried out as given in the guidelines of WHO. [26]

High-performance liquid chromatography

The fruit, leaves and bark of Z. armatum were washed separately and shade dried. The dried parts were ground to fine powder. The extraction was carried out using 2 g of powdered material with 50 mL of methanol under 80 kHz, 40°C in ultrasonic extraction device for 35 min and repeated thrice. The extracts were collected and filtered; the filtrate was dried at 40°C under reduced pressure in a rotary evaporator.

High performance liquid chromatography (HPLC) with autosampler (PerkinElmer series 200, USA), UV-visible detector, column (SUPELCO) C18, 25 cm × 4.6 mm, particles with diameter 5 μm was used. Samples were analyzed using TotalChrom Workstation software, version 6.3 with liquid chromatography instrument control was used for analysis. Gallic acid (GA) was purchased from Sigma (MO, USA) and Rutin (RU) from Fluka (Germany). All the HPLC grade solvents for elution and sample preparation were purchased from Merck (Germany). Standard stock solutions of three phenolic compounds and sample solutions of extracts were prepared in methanol and filtered through 0.45 μm membrane filter (Millipore).

The gradient elution of solvent A (water-acetic acid [25:1 v/v)]) and solvent B (methanol) had a significant effect on the resolution of compounds. Solvent B was increased to 50% in 4 min and subsequently increased to 80% in 10 min at a flow rate of 1.0 mL/min. Detection wavelength was 280 nm and 360 nm. Injection volume was 10 μl for sample and references each. Run time was 14 min.[3334]

RESULTS

Macroscopic characters

Leaves, stem bark and fruit of Z. armatum were evaluated on morphological parameters. The observations for size, color, shape, texture, fracture, taste and odor were noted for leaves, bark and fruit and are tabulated in Table 1. Similarly, the powdered drug was also evaluated organoleptically [Figure 1].

Table 1

Organoleptic evaluation of Zanthoxylum armatum leaf, bark and fruit

Figure 1

Zanthoxylum armatum leaves, fruit and bark (with and without spines)

The transverse section of a leaf of Z. armatum shows dorsi ventral characters. The important tissues in the midrib regions are as follows:

The midrib is hemispherical at the dorsal side and convex at the ventral side. The less distinct epidermis is composed of single layer of cells and is covered with a thin cuticle. Collenchyma consists of 2–4 layers of cells above the lower epidermis and very few cells are visible below the upper epidermis. Very prominent large spongy cells of pith are located at the center. Pith is surrounded by vertical layers (≥30) of small, spherical cells (8–10 in each layer) of xylems. Xylems are surrounded by crescent-shaped phloem and schlenrenchyma. Schlenrenchymatous cells (layers of pericyclic fibers) are small and irregularly arranged. In the mesophyll tissue, spongy parenchymatous cells are loosely arranged, and not much distinct and the palisade is consists of compact, elongated, and large cells [Figure 2].

Figure 2

Microscopic characters of Zanthoxylum armatum leaf (TS)

The leaf stomata of Z. armatum are of the anomocytic type with lip-shaped guard cells. The stomatal index, vein termination and vein islets number are tabulated. The palisade cells of varying shapes and sizes are noted [Table 2].

Table 2

Quantitative microscopic and physicochemical parameters for Zanthoxylum armatum leaf, bark and fruit

The leaf veins are prominent to tertiary level. The vein islets are random in directions and dimensions. The vein islets are fairly prominent with thick boundaries. The vein islets are prominent distinct, wide, polyhedral and randomly oriented. The boundaries of the islet are thick. The vein terminations are arboreous. One islet mostly contains one vein termination [Table 2 and Figure 3].

Figure 3

Venation pattern of Zanthoxylum armatum leaf

The powder drug microsocopy of leaves shows the following diagnostic and anatomical features: Lip shaped stomata with closed guard cells of anomocytic type; the SEM of epidermis shows the arrangement of stomata and a group of fibers. The powder drug microscopy of fruit shows oil cells of testa; oil secretory rounded parenchymatous cells of fruit, distinct spiral vessels and surface view of epicarp. The powder drug microscopy of bark revealed the following anatomical features: A cross-sectional view of vascular vessels, phloem parenchyma lying in between the group of fibers, square shaped cork cell and fibers in groups [Figure 4].

Figure 4

Scanning electron microscopic images of powdered drug samples of leaf (l), fruit (f) and bark (b) of Zanthoxylum armatum plant. LA: Stomata with closed guard cells. LB: Leaf surface showing the arrangement of stomata liquid chromatography: Group of fibers LD: Light microscope image of epidermis showing arrangement of stomata. FA: Vascular Spiral vessel FB: Oil secretory rounded parenchymatous cells of fruit. FC: Epicarp in surface view. FD: Light microscope images of oil cells of test. BA: Vessels in bark, a cross view BB: Group of fibers surrounding the phloem parenchyma. BC: Group of broken and loose fibers BD: Light microscope image of cork cells in surface view

Determination of physicochemical parameters

The total ash values, for leaves, bark and fruit are determined and given in percentages. Water soluble ash values, which indicate the presence of cellulosic substances, are also calculated for leaves, fruit and bark. High acid insoluble ash values for leaves, fruit and bark, indicate the presence of silicacious substances [Table 2].

Significant amount of moisture was found in air-dried material of fruit. Similarly, leaves and bark showed moderate values of LOD. Therefore, care should be taken while storing fruits of Z. armatum especially in powdered form [Table 2].

Various solvents like methanol, ethyl acetate, chloroform, and hexane are used to determine the extractive values, which are important quality control parameters for herbal drugs. As expected, the extractive values were more with polar solvents e.g., methanol and decreased as the polarity decreases, e.g., hexane. In case of bark, chloroform extractive value was significant as compared to ethyl acetate [Table 2].

Fluorescence analysis is also an important tool for the determination of constituents in herbal drugs and it provides an idea about their chemical nature. The powder drug analysis was performed when treated with various chemical reagents and observations were made in visible light and UV light of short and long wavelengths [Table 3].

Table 3

Fluorescence analysis of powdered drug of Zanthoxylum armatum leaf, bark and fruit in ordinary light and UV light after treated with different reagents

Preliminary phytochemical and histochemical screening

Qualitative analysis of leaves, fruit and bark of Z. armatum showed the presence of various phytochemicals. More than one test was employed in the case of alkaloids, flavonoids, tannins, saponins and steroids. Alkaloidal contents are detected in leaves, fruit, and bark. Analysis showed the presence of flavonoids, saponins and terpenoids in respective tests. Condensed tannins were detected in all three samples. Steroids are only detected in leaves and anthraquinones were absent in the bark [Table 4].

Table 4

Phytochemical detection of leaf, bark and fruit of Zanthoxylum armatum

Histochemical analysis showed the presence of tannins, starch, mucilage, fats, fatty oils, volatile oils and resins, lignified cell walls, and calcium carbonate. The cellulose cell walls were detected in bark and fruit [Table 5].

Table 5

Observations for various histochemical detection tests for leaf, bark and fruit of Zanthoxylum armatum

High performance liquid chromatography

Rutin, GA and other related flavone glycosides can be used as possible quality control indicators in herbal products. The HPLC qualitative analysis carried out showed the presence of RU and GA in methanol extracts of Z. armatum bark and leaves when the absorbance was set at 360 nm and 260 nm respectively. Similarly, the Z. armatum fruit extract also showed the presence of RU at 360 nm of absorbance [Figure 5].

Figure 5

High performance liquid chromatography chromoatogrpah of Zanthoxylum armatum bark, leaf and fruit with standard phenolic compounds (rutin and gallic acid) at different wavelengths

DISCUSSION

This study is an attempt to establish, the diagnostic characteristics of Z. armatum. These results can be employed as suitable quality control measures to ensure the quality, safety and efficacy of this herbal drug material. The parameters studied here are useful to identify and authenticate the traditionally important medicinal plant Z. armatum and this will prove helpful in the preparation of herbal monographs and pharmacopoeial standards as emphasized by WHO.

Because the safety and efficacy are the ultimate goals, to ensure the reproducible quality of the herbal drugs, the exact identification and quality assurance of the raw material is essential.[35]

Mostly the herbal materials are supplied to the market is shrunken, twisted, rolled and deformed and without trade name and proper identification. So, such drugs can easily adulterated or substituted.

The application of pharmacognostic protocols such as macromorphology, micromorphology, organoleptic tests, ash value, histochemical studies and UV fluorescence study will help in identifying genuine drugs because these tests result in specific results for a particular drug.

Microscopy also plays an important role in drug identification. The importance of epidermal characters, in general, are widely recognized in taxonomic considerations and in many cases these are successfully used in the identification of taxa at genus as well as species levels.[36] Similarly, studies in stomata have a great taxonomic as well as pharmacognostic value in proper identification of medicinal plants.[37]

The physical parameters are almost constant for a plant therefore these are helpful in setting standards for a crude drug. Various physicochemical parameters were evaluated for the leaf, stem bark and fruit parts as mentioned in WHO guidelines. These parameters are important for detection of drug adulteration or improper handling of raw materials.[1] One such parameter is ash value, which gives an idea of inorganic composition and other impurities in a plant drug. The total ash value is also important for detection of metal, salts, and silica.[38]

There are always chances of microbial growth when the crude drug is stored for a longer period of time and the moisture content of crude drug is directly related to its stability and consequently with the shelf life of crude drug. The lower the moisture content, the higher will be the stability of that drug and chance of microbial growth will be less and vice versa.[39]

Identification of the different classes of phytochemical constituents of the plant is an important parameter, which gives an indication of the pharmacological active metabolites present in the plant.[40] Phytochemical studies on Z. armatum indicate it to be a useful plant to investigate for phytochemical and biological assays. HPLC analysis may serve as a useful data for the standardization of the drug.

The data generated from this study would help in the authentication of various parts of Z. armatum, a very important constituent of various herbal drug formulations. This may lead to easier authentication of herbal drugs procured from markets for the correct identification of the medicinal plant ingredients.