Formulation and Evaluation of Quality Parameters of Effervescent Granules from the Potent Antioxidant between Two Variants of the Adaptogenic Herb Ocimum tenuiflorum L.

Ocimum tenuiflorum L. is found throughout semitropical and tropical parts of Southeast Asia. In Nepal, O. tenuiflorum L. is popular with two variants: Krishna Tulsi consisting of purple-colored leaves and Sri Tulsi consisting of green-colored leaves. O. tenuiflorum L. is considered the queen of herbs and is a traditionally and clinically proven medicinal herb for its application and efficacy. However, no commercial pharmaceutical preparations of O. tenuiflorum L. are available using effervescent vehicles. Therefore, the present study aimed to compare the antioxidant activity of leaves from the two varieties of O. tenuiflorum L. and formulate and evaluate the quality parameters of effervescent granules of the potent extract. The antioxidant activity of O. tenuiflorum L. ethanolic extracts was evaluated by DPPH radical scavenging assay at 1, 10, and 100 µg/mL concentrations, where ascorbic acid was used as the positive control. The antioxidant activity of purple-leafed O. tenuiflorum L. was found to be higher than that of green-leafed O. tenuiflorum L. Therefore, effervescent granules of the ethanolic extract of purple-leafed O. tenuiflorum L. were formulated using the pharmaceutical excipients tartaric acid, citric acid, and sodium bicarbonate and the quality parameters of the granules were evaluated. The formulated granules met the quality parameters assessed from the angle of repose, bulk density, tapped density, Carr's Index, Hausner's ratio, effervescent cessation time, and stability studies. Thus, the formulated effervescent granules of O. tenuiflorum L. can be used for therapeutic purposes or as a functional food.


Introduction
From past to present, the use of plants has always maintained its importance. Te most striking part of these usage areas is pharmacology [1][2][3]. Recently, lifestyle-related chronic diseases have become the prevailing causes of global morbidity and mortality, many of which could be managed through Ayurveda with its primary focus on the regular consumption of adaptogenic herbs and healthy lifestyle practices. Te consumption of adaptogenic herbs periodically could enhance the body's capacity to maintain balance amid a variety of stressors [4].
Te consumption of O. tenuiforum L. on a regular basis is said to prevent disease, promote general health, wellbeing, and longevity, and help the body and mind cope with a wide range of chemical, physical, infectious, and emotional stresses and restore physiological and psychological function [4,[8][9][10][11]. O. tenuiforum L. can also provide luster to the complexion and sweetness to the voice and foster beauty, intelligence, stamina, and a calm emotional disposition [8][9][10][11]. O. tenuiforum L. has long been consumed as tea herbs/a cuisine material in Vietnam and other Southeast Asian countries [5]. Te best known active compounds that have been identifed and extracted from O. tenuiforum L. are eugenol, the major component of essential oil, and ursolic acid [9]. It has been reported that the leaves of this plant contain essential oils composed of limonene, borneol, copaene, caryophyllene, and elemol, favonoids (apigenin, luteolin, apigenin-7-O-glucuronide, orientin, and olludistin), triterpenoids (oleanolic and ursolic acid), polyphenols, and tannins [5,14].
In the context of Nepal, two varieties of O. tenuiforum L. are available. One of the varieties commonly known as Krishna Tulsi consists of purple-colored leaves and another variety known as Sri Tulsi comprises green-colored leaves. Te antioxidative activity of O. tenuiforum L. has been already proved, but the activity of the two varieties has not been compared. Tough O. tenuiforum L. is widely used traditionally to treat various disorders and its clinical efcacy has been proven, it has not been available in appropriate dosage forms.
Te efervescent granules proved to be a promising vehicle for plant extracts due to their simplifed processing, easy administration, rapid disintegration, good fow properties, and low cost [20]. By considering the therapeutic potentials of O. tenuiforum L. and the advantages of effervescent formulation, the study aimed to evaluate the antioxidant activity of leaves of green-leafed and purple-leafed varieties of O. tenuiforum L. and develop the efervescent granules of the potent extract and evaluate the quality parameters of the formulated granules.

Extraction.
Te plant samples were extracted using the maceration method. Te ground powder (100 g) of each plant material was extracted with ethanol (2 × 700 mL) at room temperature for 24 hours. Te extracts were then concentrated to dryness under a rotary evaporator. Te extraction yield value of the plant extracts was calculated using the following formula: % yield � weight of extract obtained weight of crude drug taken × 100. (1)

DPPH Radical Scavenging
Assay. DPPH radical scavenging activity was measured according to the method described by Baral and Basnet [21] with slight modifcations. Two mL of ethanolic solution of each extract at various concentrations (1 µg/mL, 10 µg/mL, and 100 µg/mL) was mixed with 2 mL of ethanolic solution of DPPH (approx. 60 µM) and left for 30 minutes at room temperature. Te antioxidative activity of extracts corresponding to the scavenging of DPPH radical was measured at 517 nm by the absorbance of UV spectrophotometer.
Radical scavenging activity (%) � control absorbance − extract absorbance control absorbance × 100, where control was the test solution without extract. Ascorbic acid was used as the positive control.

Formulation of Efervescent Granules.
Efervescent granules of the ethanolic extract of the purple-leafed O. tenuiforum L. were formulated by the wet granulation method described by Xiang-Zhou and Sheng [22] and Forman et al. [23] with some modifcations. According to Forman et al. [23], the best efervescent vehicle ratio of citric acid, tartaric acid, and sodium bicarbonate is 1 : 2 : 3.44. So, to prepare the formulation, 12 g citric acid, 24 g tartaric acid, 41.28 g sodium bicarbonate, and 2.5 g O. tenuiforum L. extract were triturated into a fne powder, and then a sufcient quantity of ethanol was added to make a damp mass and passed through sieve no. 10 to obtain granules, and fnally, they were dried in a hot air oven at 40°C and packed in an airtight container.

Evaluation of Formulated Herbal Efervescent Granules.
Angle of repose, bulk density, tapped density, Carr's Index, and Hausner ratio were calculated according to Aulton and Kelvin [24], and efervescent cessation time of the formulated efervescent granules was calculated using the method described by British Pharmacopoeia [25], and stability studies were performed according to the procedure explained by Shet et al. [26].

Angle of Repose.
Te angle of repose of the formulated granules was measured using a fxed funnel method. A funnel was ftted into a stand with its tip at 2.5 cm above a graph paper that is placed on a fat horizontal surface. Te efervescent granules were carefully poured through the funnel until the apex of the conical pile touched the tip of the funnel. Ten, the radius of the base of the conical pile was measured. Te angle of repose was fnally calculated using the following formula: where θ � angle of repose, h � height of the pile cone, and r � radius of the cone base of the pile.

Bulk
Density. Formulated granules of 15 g were introduced into a dry 100 mL cylinder, without compacting. Te level of the granules was carefully marked without compacting and the unsettled apparent volume, Vo, was read. Ten, the bulk density was calculated using the following formula: where ρb � apparent bulk density, M � weight of the sample, and V � apparent volume of powder.

Tapped Density.
After carrying out the procedure as given in the measurement of bulk density, the cylinder containing the sample was tapped 500 times initially, followed by additional taps of 750 times until the diference between succeeding measurements was less than 2% and then tapped volume, Vf, was measured to the nearest graduated unit. Te tapped density was calculated, in gram per mL, using the following formula: where ρtap � tapped density, M � weight of sample, and Vf � tapped volume of powder. Te Scientifc World Journal

Carr's Index (%). Carr's Index (compressibility index)
is a measure of the propensity of a powder to be compressed. It was determined from the bulk and tapped densities using the following formula: where ρtap � tapped density and ρb � bulk density.

Hausner Ratio.
Hausner ratio is considered as the indirect index of ease of powder fow. It was calculated using the following formula: where ρtap � tapped density and ρb � bulk density.

Efervescent Cessation Time.
A volume of 200 mL of distilled water was taken in a beaker, one dose of efervescent granules (3 g) was poured into the beaker, and efervescent cessation time and efervescent production were observed.

Stability Studies.
For stability study of the formulation, efervescent cessation time was carried out after the 1 st month, 2 nd month, and 3 rd month of formulation at normal room temperature, i.e., at 27 ± 2°, and accelerated temperature, i.e., at 40 ± 2°, and also the granules were examined visually for any changes in color, odor, and texture. Table 1.

Extraction of Yield Value. Te percentage yield value of the ethanolic extracts of plant samples is shown in
Te extract yield percentage of the leaves of purple-leafed O. tenuiforum L. was found to be relatively higher than that of green-leafed O. tenuiforum L.

DPPH Radical Scavenging Activity.
Te percentage of DPPH radicals scavenged by ascorbic acid and O. tenuiforum L. leaf extracts at 517 nm is shown in Figure 2. Te standard drug, ascorbic acid, was able to scavenge 99% of the DPPH radical at 100 µg/mL concentration, whereas the ethanolic extracts of purple-leafed O. tenuiforum L. and green-leafed O. tenuiforum L. were able to scavenge 91% and 89% of the DPPH radicals, respectively. Similarly, Figure 3 shows IC 50 values of ascorbic acid and O. tenuiforum L. leaf extracts.

Formulation of Herbal Efervescent Granules.
Te antioxidative activity of the ethanolic extract of purple-leafed O. tenuiforum L. was found to be higher than that of greenleafed O. tenuiforum L. Terefore, the ethanolic extract of purple-leafed O. tenuiforum L. was selected to prepare the efervescent granules. Te formulated efervescent granule is shown in Figure 4.

Discussion
Plants have been found as an important source of drugs. A large number of the world's populations, especially in developing countries, depend upon medicinal plants as an alternative and complementary drug therapy for various ailments [14,27,28]. According to the WHO, more than 60% of the world population still relies on herbals and herbal-originated medicaments for treating short and chronic ailments, and due to their widespread usages, many domestic and multinational pharmaceutical companies are inducted into the production of various herbal formulations [29]. Te acceptability of herbal formulations by the public is increasing nowadays due to their afordability, efectiveness, and less toxicity. Te efectiveness and therapeutic efcacy are major concerns for any type of formulation. Particularly, in addition to the above concerns, built-in quality and product standards are also key concerns for herbal formulations [30][31][32].
O. tenuiforum L. is widely used in a number of traditional medicines. It has been found to be very efective, safe, and inexpensive in relation to its availability. Due to its proven medicinal values, O. tenuiforum L. has been considered as an important plant among other herbs [33]. Terefore, O. tenuiforum L. was selected for the present study to evaluate its antioxidant activity. In recent years, the search of antioxidant is of prime concern because of their use in therapy for cardiovascular diseases, ageing, cancer, arthritis, and various other diseases arising due to oxidative stress and cellular damage [34,35]. Te antioxidant activities of the prepared plant extracts were investigated by the DPPH radical scavenging assay, a very simple and reliable method to evaluate the antioxidant activity of plant extracts. DPPH forms a stable molecule when it accepts an electron or a hydrogen atom and thus can be used in the determination of radical scavenging activity of natural products [36,37]. Free radicals, which are present in our biological systems, may oxidize all the biological molecules such as proteins, nucleic acid, and lipids and initiate  degenerative diseases [38,39]. Antioxidants can scavenge the free radicals generated within the body and also play a great role in inhibiting the chain reaction of lipid peroxidation [20,36]. In the present study, the antioxidative properties of both standard drug and plant extracts increased with increasing concentration.
Values for the angle of repose ≤30°usually indicate a free fowing material and angles ≥40°suggest a poorly fowing material, 25-30 show excellent fow properties, 31-35 show good fow properties, 36-40 show fair fow properties, and 41-45 show passable fow properties [24]. In this study, the formulated granules had an angle of repose 32.28°, so it can be concluded that the formulated efervescent granules have good fow properties. Lower Hausner ratio (<1.25) indicates better fow properties than higher ones, between 1.25 and 1.5 showing moderate fow properties and more than 1.5 poor fow [24]. Te prepared formulation had Hausner ratio of 1.05, so it also indicates that the granules have good fow properties.
Pandey et al. [40] reported that the angle of repose of formulated efervescent granules of Martynia annua was 33.02, and bulk density (ρb) and tapped density (ρtap) were 0.55 and 0.71, respectively; the compressibility index (Carr's Index) was 22 and the Hausner ratio was 1.29, which showed its moderate fow property, and the efervescent cessation time was 2-3 min, whereas in this study, all the values were found to be less than the values in the previous study and showed better fow properties and efervescent cessation time.
From stability studies, it was found that the formulated granules had similar efervescent cessation time and visual characteristics compared to initial parameters at both    Te Scientifc World Journal normal room temperature and accelerated temperature, i.e., at 40 ± 2°. Tis means that the formulated granules were found stable even after 3 months at both the conditions. Te stability of the solid dosage form is higher compared to its liquid preparations as the potential degradation of active ingredients in aqueous media, microbial contamination, and the associated disruption of its shelf life are prevented. Te efervescent granule should be dispersed in water to prepare a solution for its administration, which will provide an improvement in taste, solubility, and bioavailability of the herbal extract [41].

Conclusion
Two varieties of O. tenuiforum L., green-leafed and purpleleafed, were selected to evaluate the antioxidant activity. Te antioxidant activity of purple-leafed O. tenuiforum L. was found higher than that of green-leafed O. tenuiforum L. Te formulated granules with the purple-leafed ethanolic extract showed good fow properties as depicted from the angle of repose, bulk density, tapped density, Hausner ratio, and compressibility index. Te efervescent cessation time was found within the limit and the formulated granules also passed the stability test. Tus, the efervescent granules can be a promising vehicle for plant extracts due to their simplifed processing, easy administration, rapid disintegration, and good fow properties.

Data Availability
Te data used to support the fnding of this study are available upon request from the author.

Conflicts of Interest
Te author declares that there are no conficts of interest regarding the publication of this paper.