INTRODUCTION

Green technology and alternative eco-friendly products are brand new to several.¹ New lifestyle changes cause many perilous drawbacks, which opens a gateway for the search of new resolves. Thus, at present, the term “Green” has become much popular. The major area under “Green” consideration will be the field of traditional or herbal medicine. Herbal medicines are safe remedies for various human ailments, given their minimal side effects and low-cost treatments. Thus, there is wide acceptance of herbal medicinal system. The quality measurements of herbs are challengeable streams, where validations of herbs are more important than their usage. Adulterations are drawbacks in the field of herbal medicine, as they cause inconsistences in quality and safety of herbal preparations. This has opened a new approach to validate the quality assurance of herbs. The collection of plant materials, authentication of specimens, analysis, and formulation of drugs are the majorly value chain toward the discovery of safer natural drugs. Here in, an attempt was made to evaluate the pharmacognostic characters of the medicinally important genus Memecylon fruits.

Memecylon is the genus of the family Melastomataceae. There are 289 species of shrubs and trees widely distributed in tropical regions. In India, about 40 species were reported and 21 among them are endemic to the country.² Memecylon species are difficult to identify due to the fact that their morphological similarities are some what confusing. Memecylon umbellatum and Memecylon randerianum are common species found in the Western Ghats. Another selected species for the present study is Memecylon grande, which is present in the Western Ghats and dry deciduous forests. Previous reports have revealed that these three species of Memecylon have potent medicinal activity.^3,4 M. umbellatum possesses elliptic-lanceolate leaves with umbellate inflorescence and yellow berries. M. randerianum possesses ovate-oblong leaves and blueberries, while M. grande has ovate-lanceolate leaves with brownish blackberries.

Many systematic studies and new records are available on the genus Memecylon; however, evaluations of micromorphological characters are trivial. Scanning electron microscopy (SEM) analysis is the best way to analyze the surface features of the samples. In taxonomic identification, analysis of vegetative and reproductive characters through SEM is an effective method.⁵ The functional purity of plant samples is essential for pharmaceutical trails. In this present study, purity of sample was analyzed by powder microscopy, SEM-energy dispersive X-ray (EDX), and inductively coupled plasma-mass spectrometry (ICP-MS) techniques. Powder microscopy acts as an effective tool for authentication plant materials.⁶ SEM-EDX and ICP-MS are effective analytical methods for analyzing metal nano- and micro-particles in food products, plant, or environmental.⁷ The backbone behind the performance of plants always hinge on the presence of bioactive metabolites. In majority of pharmaceutical studies, phytochemical analysis is a crucial step. Preliminary phytochemical analysis gives an insight on the phytochemical constituents present in plant extracts. It comprises qualitative and quantitative analysis. It provides valid information regarding the presence or absence of bioactive compounds in plant species. A plethora of biochemicals contribute to the specific bioactivity of plants, such as antimicrobial, antioxidant, and anticancer properties, among others.⁸ The present study also emphasizes the evaluation of the antioxidant potential of Memecylon fruit extracts. Antioxidants are free radical scavengers that neutralize oxidative stress induced by reactive oxygen species (ROS). Furthermore, ROS can disrupt normal cellular mechanisms and lead to severe pathological conditions and diseases, such as cancer, neurological disorders, atherosclerosis, hypertension, ischemia, and diabetes.⁹ The present study focused on the worth and utility of micromorphological characters and functional purity of fruit samples as an additional source of information in systematic studies of Memecylon.

MATERIALS AND METHODS

Plant material

Fruits of M. grande, M. randerianum, and M. umbellatum were chosen for the current study. Ripened mature fruits of the three species were collected from various parts of Kerala, India. The collected fruits were identified by Dr. A. K. Pradeep Assistant Professor, Angiosperm Taxonomy Division, Department of Botany, University of Calicut, Kerala.

SEM analysis

SEM analysis was performed using ZEISS Gemini SEM 300 machine. Samples were prepared on a carbon-coated copper grid. The technical features of Gemini SEM 300 are given below.

Gemini SEM 300 with resolutions: 0.6 nm @ 30 kV (STEM), 0.7 nm @ 15 kv, 1.2 nm @ 1 kv, and 1.1 nm @ 1kV TD. Inlens BSE resolution: 1.2 nm @ 1 kV. Resolution in variable pressure mode (30 Pa): 1.4 nm @ 3 kV and 1.0 nm @ 15 kv. Acceleration voltage: 0.02 - 30 kV. Probe current: µ3 pA-20 Na. Magnification: 12-2,000,000.

SEM-EDX analysis

SEM-EDX analysis was done using Octane plus with Gemini 300/EDS. The active area selected for the present study was 30 mm².

Powder microscopy

The fruit powder characteristics of Memecylon was analyzed under bright field microscope. The powder of the sample was treated with 4% KOH and mounted in glycerine on clean slides and the powder characters were imaged using Nikon ECLIPSE E200 trinocular microscope attached with Zeiss AxioCam Erc5s digitalcamera.

ICP-MS analysis

ICP-MS analysis was performed using Agilent 7800 ICP-MS with Integrated Sample Introduction System (ISIS3) and SPS4 autosampler. It has astandard torch of 2.5 mm-diameter injector, as well as Ni sampler and Ni skimmer cones.

Preliminary quantitative phytochemical analysis

Alkaloid content

The method of Shamsa et al.¹⁰ was followed for the determination of total alkaloid content of the selected plant material. In brief, 2 N HCl solution was prepared and 1 mg of the plant sample was dissolved in it and filtered. Phosphate buffer (pH 4.7) was prepared and 5 mL of it and 5 mL BCG solution was added and the mixture was shivered with 1, 2, 3, and 4 mL of chloroform. The chloroform layer containing alkaloids was separated. Caffeine was used as a standard. Absorbance of the solution was read at 470 nm against a blank. Alkaloid content of the sample was calculated and expressed as mg of caffeine equivalents.

Flavonoid content

Aluminium chloride colorimetric assay is a typical method for the determination of flavonoid content.¹¹ 4 mL of distilled water was put in aflask and 1 mL of the extracts was added into it. 0.30 mL of 5% NaNO₂ and 0.3 mL of 10% AlCl₃ were mixed in the flask at five minutes intervals. Again, after five minutes incubation, 2 mL of 1M NaOH was taken and the final volume of 10 mL was attained using distilled water. Finally, absorbance was read at 510 nm. The total flavonoid present in the extract was calculated and expressed as mg quercetin equivalents (QE).

Phenolic content

Phenolic content of the sample was assayed by Folin Ciocalteu assay.¹² 1 mL of the extracts was put in a 25 mL flask containing 9 mL of distilled water. 1 mL of Folin-Ciocalteu phenol reagent was mixed in the flask and shaken. At 5 minutes interval, 10 mL of 7% Na₂CO₃ solution was added and the solution was kept for 90 minutes at room temperature. Absorbance was read at 550 nm against the reagent blank. Gallic acid was taken as the standard. The phenolic content was expressed as mg of Gallic acid equivalents.

Terpenoid content

Presence of terpenoid of the plant samples was estimated by the method of Ghorai et al.¹³ The reaction solution contains an aliquot of the extract and few drops of chloroform and H₂SO₄. Absorbance is calculated at 538 nm against the blank. Linalool was taken as the standard. The terpenoid content of the extract was calculated and expressed as mg linalool equivalents (QE).

Antioxidant activity

The antioxidant potential of Memecylon fruits was documented on the basis of free radicals produced by various substrates, such as 2,2-diphenyl-1-(2,4,6-trinitrophenyl) hydrazyl (DPPH), Fe³⁺-ascorbate-EDTA-H₂O₂ system, sodium nitroprusside, and potassium ferricyanide.

DPPH radical scavenging assay

The protocol of Chang et al.¹⁴ was followed for the determination of DPPH free radical scavenging activity of Memecylon fruit extract. DPPH is a free radical that reacts with antioxidant agents and gets reduced to DPPH-H. The pink colored DPPH turns yellow when scavenged by antioxidants. The color change indicates the scavenging latent of the antioxidant substances. Reference was selected as ascorbic acid [10 mg/mL dimethyl sulfoxide (DMSO)]. Different volumes of the extracts [1.25-20 µL (12.5-200 µg/mL)] from a stock concentration of 10 mg/mL was taken and made up to 20 µL with DMSO and 1.48 mL of DPPH (0.1 mM) solution. The control was a test compound-free solution. A dark condition of 20 minutes was maintained for the reaction mixture at room temperature. Then, the scavenging potential was read at 517 nm. Percentage inhibition of DPPH free radical by the extracts was calculated as follows:

$\% \mathrm{inhibition} =\frac{\mathrm{control}–\mathrm{test}}{\mathrm{control}} \times 100$

Hydroxyl radical scavenging activity

Hydroxyl radical scavenging potential was analyzed according to the method described by Kunchandy and Rao.¹⁵ Different concentration of the samples (125-2000 µg/mL) from a stock solution of 10 mg/mL was mixed with 500 µL reaction mixture [2-deoxy 2-ribose (2.8 mM), FeCl₃ (100 µm), EDTA (100 µm), H₂O₂ (1.0 mM), ascorbic acid (100 µm) in KH₂PO₄-KOH buffer (20 mM pH 7.4)] and made up to 1 mL. The control was a solution devoid of the test material. The test sample tubes were kept at 37°C for 1 hour. 1 mL of 2.8% TCA and 1 mL of 1% aqueous TBA was mixed in the reaction tubes and allowed to stay for 15 minutes at 90°C for the color change. Absorbance was read at 532 nm against a blank. Gallic acid (10 mg/mL DMSO) was used as reference. Percentage inhibition of hydroxyl radical by the extracts was calculated as follows:

$\% \mathrm{inhibition} =\frac{\mathrm{control}–\mathrm{test}}{\mathrm{control}} \times 100$

Nitric oxide radical scavenging activity

Determination of nitric oxide radical scavenging activity of Memecylon fruit extracts was performed according the method described by Kumaran and Karunakaran.¹⁶ Sodium nitroprusside (5 m/mol/L^-1) in phosphate buffered saline solution (pH 7.4) was added into various concentrations of extracts (125-2000 µg/mL) from a stock solution. The reaction mixtures were then incubated at 25°C for 30 minutes. A control was also prepared and incubated accordingly. After 30 minutes incubation, 1.5 mL of the reaction mixtures were removed and 1.5 mL of Griess reagent (1% sulphanilamide, 2% phosphoric acid, and 0.1% N-1-naphthylethylenediamine dihydrochloride) was added. Absorbance was read at 546 nm. Gallic acid (10 mg/mL DMSO) was used as a reference compound. Percentage inhibition of nitric oxide radical by the extracts was calculated as follows:

$\% \mathrm{inhibition} =\frac{\mathrm{control}–\mathrm{test}}{\mathrm{control}} \times 100$

Superoxide radical scavenging activity

Superoxide radical scavenging assay was performed according to the method described by Valentão et al.¹⁷ A concentration range of (125-2000 µg/mL) was taken from a stock solution (10 mg/mL). The reaction tube contained 0.05 mL of riboflavin solution (0.12 mM), 0.2 mL of EDTA solution (0.1 M), and 0.1 mL of nitro blue tetrazolium solution (1.5 mM). 2.64 mL of phosphate buffer (0.067 M) was added to the reaction mixture. Absorbance was read at 560 nm at two illumination times of 5 minutes and 30 minutes, respectively. Ascorbic acid (10 mg/mL DMSO) was used as reference. Percentage inhibition of superoxide radical by the extracts was calculated as follows:

$\% \mathrm{inhibition} =\frac{\mathrm{control}–\mathrm{test}}{\mathrm{control}} \times 100$

RESULTS AND DISCUSSION

SEM is a high-resolution surface imaging technique that uses an electron beam with superior magnification power and depth of field precision. The seed capsule micromorphology and the entire seed morphology were studied by SEM analysis. The difference in electron emission in different areas provides the surface topography of the material. In this study, all the selected species showed distinct morphological patterns. Surface morphology is an important index in taxonomic identification.¹⁸ SEM analysis of Memecylon fruits is a novel report. In the case of M. grande, fruits show a colliculate pattern, with a seed capsule of 6.2 mm, and the seed surface possesses a tuberculate pattern, with a width of 5.8 mm (Figure 1 A1, B1, C1 and D1). SEM analysis revealed that the seed capsule of M. randerianum a ruminate reticulate type pattern (Figure 1 A2, B2, C2 and D2). The width of the capsule was 4.2 mm. The seed surface of M. randerianum was of a reticulate pattern, with a width of 3.5 mm. M. umbellatum seed capsule possesses a smoothened pattern, with a width of 5.6 mm, and its seed surface showed a wrinkled pattern, with a width of 3.81 mm (Figure 1 A3, B3, C3, and D3). The characteristic surface morphology is a useful tool in the identification process.

EDX is an effective way of analyzing elemental compositions at tiny level. In this study, SEM was coupled with an energy dispersive system with an electron probe for X-ray microanalysis. The surfaces of the specimens were evaluated by SEM-EDX analysis and this gave a clear picture of the elemental composition of the test sample.¹⁹ The elemental composition of M. grande fruits show that nitrogen content was 91%, while that of other elements were as follows: Phosphorus (3.10%), potassium (1.53%), iron (1.41%), magnesium (0.87%), and sodium (0.55%) (Figure 2). In the case of M. randerianum fruit, nitrogen was the prominent element, with 93% of the weight. Copper (0.49%), cobalt (0.90%), zinc (0.09%), sodium (0.03%), magnesium (0.22%), phosphorus (4.01%), potassium (1.15%), and calcium (0.09%) were the composition of other elements (Figure 3). M. umbellatum fruit also possesses an elevated amount of nitrogen (93%) and the other elements were in trace amount: Phosphorus (3.4%), potassium (1%), copper (0.95%), magnesium (0.67%), cobalt (0.34%), and iron (0.22%) (Figure 4). This finding indicates that Memecylon fruits are reservoirs of essential elements and can be exploited in the pharmaceutical or nutritional field.

In Ayurveda, 90% of the preparations are plant-based, implying that the worthwhile usage of herbal medicines are promising remedies for diverse human diseases. In most Ayurvedic preparations, the powdered samples of plant parts are used. Therefore, the authenticity of the powdered sample is very important. Powder microscopy is a simple and easier method to analyze powdered samples and is an essential step in pharmacognostic evaluation of plant samples. Microscopic techniques examine the structural and cellular features of herbs, so as to determine their botanical origin. Microscopic evaluation is now an indispensable tool for the identification of medicinal herbs and is among the important parameters in this modern time.²⁰ Here in, the powdered samples of Memecylon fruits were characterized by their microscopic characters. The powdered M. grande fruits was brown in color, odorless, and slightly astringent (Figure 5). The characters found in the powders were epicarp cells, parenchyma cells with starch grains from mesocarp, stone cells from mesocarp, sclereids from endocarp, vessels with spiral and annular thickenings, and rosette crystals. In the case of M. randerianum fruits powder, it is brown-colored, odorless with a characteristic taste (Figure 6), and contains epicarp cells, mesocarp parenchyma cells, stone cells, sclereids from endocarp, tracheids, fiber bundles, and rosette crystals. The same brown colored powder was also seen for M. umbellatum fruits (Figure 7), which showed characteristics, such as epicarp cells, pitted parenchyma cells from mesocarp, stone cells, sclereids, spiral vessels, fibro-sclereids, and rosette crystals. These characters can be used to identify authenticated plant specimens in Ayurvedic preparations. Therefore,we can easily identify the botanical origin of the plant specimen and clearly distinguish the presence of adulterants or allied species. Microscopic evaluation of M. umbellatum leaves was done according to the method described by Killedar et al.⁶ and the presence of lignified xylem with well-defined xylem fibers, vessels, and parenchyma was found. The presence of phloecentric vascular bundles surrounded by endodermis and crystal sheath was also reported. The powder microscopic analysis confirmed that the botanical origins of the plant samples were pure and devoid of foreign particles. Therefore, this result can be used as a future reference for identification of Memecylon fruits.

During the past decades, much attention have been focused on nutritional status. We had an insight into the profound effect of micro and macronutrients on biological processes that range from whole-organism performance to cellular function. According to the classification of trace elements, group I, which consists of carbon, hydrogen, oxygen, and nitrogen, are the key building blocks of carbohydrates, proteins, and lipids. Group II includes nutritionally important minerals, such as sodium, potassium, phosphorous, chloride, calcium, magnesium, and sulfur. Trace elements, such as like copper, iron, zinc, chromium, cobalt, iodine, molybdenum, and selenium, are found in group III.²¹ Copper is important in metabolism, mainly in the proper functioning of the enzymes and its deficiency can lead to hypochromic anemia, joint pain neutropenia, hypopigmentation of hair and skin, abnormal bone formation with skeletal fragility, and osteoporosis, among others.²² Another most important element is iron, which is a prime portion of the blood cells and its deficiency is called anemia. Anemia is the second vital cause of maternal mortality in India and 20% of the mortality is directly related to anemia, while another 50% is associated with other anemic side effects. In the case of zinc, it is crucial for normal spermatogenesis and maturation, development of thymus, epithelialization in wound healing, taste sensation, and secretion of pancreas and gastric enzymes.²³

In addition to the SEM-EDX analysis, to substantiate the quality of the fruit samples in their elemental diversity, ICP-MS analysis was performed. This technique gave details of the elements present in the sample in parts per million and determination ofthirteen elements, including aluminum, arsenic, cadmium, cobalt, strontium, selenium, chromium, copper, molybdenum, nickel, lead, barium, and manganese, were done. M. grande fruits show a promising concentration of majority of the elements, except for molybdenum and lead. Lead and molybdenum concentration were found to be higher in the case of M. randerianum fruit (Table 1). The standard reference concentrations of trace elements present in adult human blood samples are considered, considering that it is essential for the standardization of drugs. Most of the detected elements show vital biological functions. Some elements are functional parts of vitamins, cofactors of enzymes, oxidative phosphorylation, fatty acids, and cholesterol metabolism. Lead is considered as a non-essential elemental part of the human body, since it has no biological or health roles. In the case of chromium, it acts as a modulator of enzymes and as an activator of fatty acid and cholesterol metabolism. It can also enhance sugar metabolism.²⁴ In the case of cobalt, it is the key factor of cobalamin (vitamin B12) and plays a vital role in the production of amino acids and neurotransmitters. Although the biological function of nickel is still somewhat unclear, nickel is found in higher concentration in RNA and is involved in protein structure.²⁵ Therefore, the biological role of these trace elements is significant in the maintenance homeostasis and avoidance of free radical proliferation induction and various human ailments.²⁶

Preliminary quantitative analysis of various phytochemicals was done following the proposed protocols. M. grande fruit was found to be rich in alkaloids, flavonoids, phenolics, and terpenoids (Table 2). All these phytoconstituents have a significant biological role. Liu²⁷ proposes that phytochemicals are non-nutrient compounds that can reduce the risk of major non-communicable chronic diseases and that are commonly found in fruits, vegetables, grains, and other plant foods. Alkaloids have a wide spectrum of pharmacological activity,²⁸ which includes antifungal, antihyperglycemic, antityrosinase, antiglucosidase, antinociceptive, and antiinflammatory activities, among others. The efficiency of bioactive products in curing several diseases, such ashyperlipidemia, atherosclerosis, and hypertension, were studied by Liwa et al.²⁹ Polyphenols and phenolic compounds are full sources of vascular vasodilators and are used to cure hypertension and cardiovascular diseases. Flavonoids possess anti-inflammatory, anticancerous, and antimutagenic activities, which have been previously reported.³⁰ Tan et al.³¹ confirms that the progressive use of phytochemicals through diets is an effective method to cure diseases. Memecylon fruit extract showed a promising antioxidant activity in four different assays. All the fruit extracts show a dose gradient scavenging activity. Among the extracts, M. grande fruit extract showed the highest free radical scavenging activity in all the assays, followed by M. umbellatum and M. randerianum fruit extracts (Figure 8, 9, 10, 11). M. grande fruit extracts showed that  highest nitric oxide scavenging activity (76.85±0.08) and the least hydroxyl radical scavenging activity (61.69±0.56). M. randerianum fruit showed the least hydroxyl radical scavenging activity (46.16±0.17). Based on the four assays performed, the most effective antioxidant extracts was that of M. grande fruit, with an IC₅₀ of 83.9195±0.14. This was followed by M. umbellatum, with an IC₅₀ of 91.1031±0.12, and M. randerianum, with an IC₅₀ of 104.178±0.13 (Table 3). Antioxidant capacity and total phenolic content are directly related.³² The quantitative phytochemical analysis justifies the high performance of these species in the antioxidant assays. M. grande fruit extract (370.28±8.36) possessed the highest phenolic content, making it a good candidate pharmaceutical agent.

Study limitations

The detailed phytochemical characterization and compound isolation can be more satisfactory in pharmaceutical applications. The present findings are useful in the pharmaceutical field, since the botanical purification of plants is the prime step in natural drug formulation. These results can be used as a future reference for the evaluation of Memecylon fruits.

CONCLUSION

Seed surface characteristics are used as diagnostic tools in taxonomic studies. Powder microscopy, SEM-EDX, and ICP-MS analysis have provided knowledge about the functional purity and elemental composition of Memecylon fruits samples. The free radical scavenging assays provide an acquaintance of a natural antioxidant source. Therefore, these findings can be effectively targeted toward the pharmacological utilization of Memecylon fruits.

The present investigation focuses on the seed morphology, pharmacognostic, phytochemical, and antioxidant potential of Memecylon fruits. M. grande, M. randerianum and M. umbellatum are the selected experimental plant materials. Powder microscopy, SEM-EDX and ICP-MS analysis were carried out to analyze the surface features and elemental composition of the Memecylon fruits. The antioxidant potential of fruit samples are analyzed by DPPH, hydroxyl, super oxide and nitric oxide radical scavenging assays. The analysis of morphological features are key diagnostic tools in taxonomic studies. In the present study SEM analysis of fruit samples gave valuable results in species identification of Memecylon. The each sample shows distinct surface morohology. In ICP-MS analysis, elemental composition of fruit samples reveals the diverse elemental presence in the sample. It may helpful in pharmaceutical field for valuable medicine preparations. In the four antioxidant assays, all the selected extracts shows promising free radical scavenging activity. So all these results pointing that selected Memecylon fruit samples become a potential contributor in the pharmaceutical field.

ACKNOWLEGEMENTS

The first author gratefully acknowledges Council of Scientific and Industrial Research, Government of India (09/043(0182)/2017-EMR-I), for the award of fellowship. The author also acknowledges Central Sophisticated Instrumentation Facility, Calicut University for providing SEM-EDX, ICP-MS facilities for the research work.

Conflicts of interest: No conflict of interest was declared by the authors. The authors alone are responsible for the content and writing of the paper.