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FORMULATION AND INVESTIGATION OF ANTIOXIDANT POTENTIAL OF O/W LOTIONS CONTAINING Tamarindus indica L. FRUIT PULP EXTRACT

The fruit pulp extracts of Tamarindus indica have been reported to possess several biological activities, especially antioxidant property which is suitable for cosmetic application. Therefore, the aims of this study were to formulate the tamarind fruit pulp extract loaded lotions (o/w emulsions), and to assess the antioxidant activity of tamarind fruit pulp extract loaded lotions. Initially, tamarind fruit pulp extracts were prepared by a solvent extraction method. The solvents used were water or a mixture of water and ethanol (water: ethanol= 1:1). Afterwards, the obtained tamarind fruit pulp extracts were subjected to lyophilization process. The tamarind fruit pulp extracts were tested for antioxidant activity by 2, 2-diphenyl-1-picrylhydrazyl (DPPH) assay to determine suitable concentrations of the extracts to be incorporated into the lotions, which were 2 and 4%w/w in the current study. Apart from the basic ingredients of the oil phase and water phase, ViscOptima SE (2%w/w) was selected as an emulsifier and a thickener of the formulations. The tamarind fruit pulp extract loaded lotions were characterized for physicochemical property and antioxidant potential. The freshly prepared tamarind fruit pulp extract loaded lotions were light brown with homogeneity and no phase separation was observed after centrifugation at 3,000 rpm for 30 min. They had weak acidic pH (4.4-5.1), considered acceptable for skin application. The loaded formulations (F1, 2%w/w) and F2, 4%w/w)) exhibited significantly higher conductivity values than that of the unloaded formulation (F0) (p<0.05). The formulations behaved as pseudoplastic flows with low viscosity. The DPPH measurement revealed that the formulations F1 and F2 had potential antioxidant activity. In conclusion, topical o/w lotions containing tamarind fruit pulp extract were successfully prepared. They had substantial antioxidant activities. As a result, tamarind fruit pulp extract loaded lotions displayed a potential use in cosmetic formulations, especially antiaging products.


Introduction
Nowadays, a variety of plant extracts has been applied in cosmetic field. Among them is the extract of Tamarindus indica (or tamarind) due to its remarkably useful biological activities.
The fruit pulp extract of tamarind is of interest since it possess antioxidant property which is promising for antiaging products. Several signs of aging such as wrinkles, hyperpigmentation and age spots are associated with oxidative stress (Saliou, Weber, Lodge, & Packer, 2014). Therefore, tamarind fruit pulp extract would have high potential in reducing or preventing these signs of aging. In the current study, tamarind fruit pulp extract was prepared from ripe tamarind fruit pulp (sour type) using water or a mixture of water and ethanol as a solvent, followed by lyophilization process. Lotions (o/w emulsion type) were selected as vehicles for the obtained tamarind fruit pulp extract owing to aesthetic appearance, good skin feeling, ease of application and suitability for acne prone skin. The two immiscible oil and water phases were emulsified by an appropriate emulsifier, here ViscOptima SE (by Croda) which could also act as a thickener and a stabilizer.
Thus, it is beneficial for formulating plant extracts. The INCI name of ViscOptima SE is sodium polyacrylate, ethylhexyl cocoate, PPG-3 benzyl ether myristate and polysorbate 20. Derived from coconut, it is in a liquid state and complied with Inventory of Existing Cosmetic Ingredients in China (IECIC) (Croda datasheet). The beaker method was employed to prepare the lotions. After mixing oil phase and water phase together, suitable amounts of tamarind fruit pulp extract based on the antioxidant activity (EC50, effective concentration of sample required to scavenging DPPH radical by 50%) were incorporated into the cool lotions and evaluated for their physicochemical properties and antioxidant activities.
The aims of the current work were to prepare tamarind fruit pulp extract using solvent extraction method, to formulate o/w lotions containing tamarind fruit pulp extract and to investigate the antioxidant potentials of the lotions containing tamarind fruit pulp extract.

Materials
The fresh ripe tamarind fruit pulps (sour type) were purchased from a local market in Na Mom district, Songkhla Province, Thailand and identified by Assoc. Prof. Dr. Juraithip Wungsintaweekul, Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Prince of Songkla University, Thailand. ViscOptima SE was a gift from Croda (Thailand) Co., Ltd (Bangkok, Thailand). Refined glycerin, cetyl alcohol, and isopropyl palmitate were supplied by Chemipan Corporation Co., Ltd (Bangkok, Thailand). L-ascorbic acid (analytical grade) was purchased from S.M. Chemical Supplies CO., Ltd (Bangkok, Thailand).
Distilled water was used in a whole experiment, except HPLC analysis (Milli-Q water was used).
All the chemicals, except L-ascorbic acid was pharmaceutical grade.

High Performance Liquid Chromatography (HPLC) Analysis
Tartaric acid, a major organic acid in tamarind fruit pulp, which has antioxidant activity (Santosh Singh Bhadoriya, Aditya Ganeshpurkar, Jitendra Narwaria, Gopal Rai, & Alok Pal Jain, 2011) was measured by HPLC method based on the analysis of Kordis-Krapez, Abram, Kac, and Ferjancic (2001). The Shimadzu HPLC system (Shimadzu, Japan) and a reverse-phase Appollo C18 column (5 µm, 4.6 x 250 mm, Alltech, IL, USA) were used for analysis. The mobile phase was 0.006 M phosphoric acid (pH = 2.1) at a flow rate of 0.8 mL/min and UV detection wavelength was 210 nm.

Preparation of Formulations
The concentrations of tamarind fruit pulp extract used were 2%w/w (5-times of EC50) and 4%w/w (10-times of EC50) according to antioxidant activity of the tamarind fruit pulp extracts (EC50 = 4.0025 mg/mL). The excipients of lotions consisted of ViscOptima SE as an emulsifier, thickener and stabilizer, cetyl alcohol (stiffening agent) and isopropyl palmitate (emollient) as oil phase, glycerin as humectant, and water as vehicle (Table 1). 1%w/v ethylhexyl glycerin and 0.2%w/v phenoxyethanol were used as preservatives. Briefly, the lotions (emulsion type) were prepared with a beaker method. Oil phase and water phase were heated until 70 o C on a water bath. Then, the oil phase was added into the water phase and stirred continously until homogenious preparation was obatined After emulsions were cool to room temperature, tamarind fruit pulp extract was added into the emulsion bases. The formulation without tamarind extract was named as F0, whreas the formulations which contained tamarind fruit pulp extract at 2%w/w or 4%w/w were named as F1 and F2, respectively.

Centrifugation Test
The stability of freshly prepared formulations were detremined with a centrifugation test at 3,000 rpm for 30 min. Then, the appearance and phase separation were observed by macroscopic observation.

Characterization of Emulsion Type
The type of the prepared emulsions were determined by a microscopic observation (light microscope) with Ponceau 4R aqueous solution and a conductivity measurement (conductivity meter, model CM-115, Kyoto Electronics Manufacturing Co., Ltd., Kyoto, Japan) for 1 day-old preaparations.

pH Measurement
The pH of the formulations were measured by a digital pH meter at room temperature (30 ± 2C), (n=3).

Viscosity and Flow Measurement
The viscosity and rheological properties of the formulations were assessed using a bobcup Brookfield rheometer (model LVDV-III Ultra, Brookfield Engineering Laboratories Inc., MA, USA) and a small adapter. The spindle SC4-31 was used. The measurements were performed at room temperature (30 ± 2C) (n=3).

Antioxidant Test
In this study, 2, 2-diphenyl-1-picrylhydrazyl (DPPH) assay was used to measure the scavening ability of both tamarind fruit pulp extracts and the lotion formulations. The DPPH test was performed using a 96-well plate and a microplate reader (NS-100 Nano scan, Hercuvan Lab Systems, UK). Firstly, the EC50 of tamarind fruit pulp extracts was determined in order to find the (1)

Statistical Analysis
Data were expressed as mean ± standard deviation (SD). Statistical comparisons were made using Student's t-test performed by SPSS statistics 20 (SPSS, Cary, NC, USA). Differences at p<0.05 were considered to be significant.

Preparation of Tamarind Fruit Pulp Extracts
The percent yield of tamarind fruit pulp 50% ethanol extract was higher than that of tamarind fruit pulp water extract, which were 61.79 and 41.60%, respectively. The two crude tamarind fruit pulp extracts had brownish-black colored and acidic smell.

High Performance Liquid Chromatography (HPLC) Analysis
The contents of tartaric acid presented in both tamarind fruit pulp extract samples were similar. In water solvent, the tartaric acid content was 24.857 mg% (w/w), while in a mixture of solvent, the amount of tartaric acid was 23.005 %mg (w/w) (p> 0.05).

Antioxidant Activity of Tamarind Fruit Pulp Extracts
According to S.S. Bhadoriya, A. Ganeshpurkar, J. Narwaria, G. Rai, and A.P. Jain (2011), several parts of tamarind such as fruits, leaves and seeds are natural sources of antioxidants which can be alternative to replacing synthetic antioxidants. Before being loaded into the formulations, screening of antioxidant activity of two tamarind fruit pulp extracts was carried out using DPPH technique. The EC50 values of tamarind fruit pulp water extract and tamarind fruit pulp 50% ethanol extract were 11.3154 mg/mL and 4.0025 mg/mL, respectively. Tamarind fruit pulp 50% ethanol extract had significantly lower EC50 than another sample (p < 0.05). However, its EC50 was markedly higher than that of a positive control, ascorbic acid (EC50 = 2.8698 µg/mL). The low EC50 value indicated that tamarind fruit pulp 50% ethanol extract had better antioxidant activity.
In a study conducted by Ćujić et al. (2016), for dried chokeberry, 50% ethanol was found to be the best solvent to obtain high yields of total phenolics and anthocyanins in comparison with the other mixed ratios between water and ethanol. The antioxidant activities of juices from Pomegranate cultivars were due to high yields of total phenolics and anthocyanins (Kalaycıoğlu & Erim, 2017).
Based on EC50 values, tamarind fruit pulp 50% ethanol extract was chosen to be incorporated into the lotion preparation.

Preparation of Formulations
All lotion formulations were successfully prepared without any phase separation after adding tamarind fruit pulp extract. This was due to the appropriate amount of ViscOptima SE used in the formulations (2%w/w). In a preliminary study, 0.5, 1 and 3%w/w of ViscOptima SE were also employed. However, phase separation happened in the case of low concentrations, 0.5 and 1%w/w, whereas too viscous lotions were obtained for 3%w/w. The macroscopic observation revealed that the formulations F1 and F2 were homogeneous with light yellow color. The prepared lotions had high aqueous proportion (water and glycerin) of about 65-69%w/w and contained the plant extract. Thus, suitable preservatives were added to inhibit bacterial growth.

Centrifugation Test
All formulations remained homogenous after 3,000 rpm centrifugation (30 min). Thus, the prepared lotions were considered stable under this experimental condition. It has been reported that the separation of two phases in the emulsion system can affect several characteristics, especially solubility of active ingredient(s) (Mhatre et al., 2015), which is not the case for the current work.

Characterization of Emulsion Type
It is generally recognized that lotions are formulations with high water content (Anton, Akram, & Vandamme, 2018). In the prepared lotions, percentages of water content started from 44.80 to 48.80 %w/w. Electrical conductivity is associated with the ability of water to pass an electrical current (Shoghl, Jamali, & Moraveji, 2016). According to conductivity values summarized in Table 2 The results of microscopic obesrvation under a ligh microscope, which are presented in Figure 1, comfirmed that the emulsion system was o/w. Red color from Ponceau 4R aqueous solution was located in the external phase (water). All formulations showed oil droplets (internal phase) with different sizes. A high speed homogenizer is recommended to achieve more homogeneous droplets.

pH Measurement
The pHs of the formulations were decreased significantly after the addition of tamarind fruit pulp extract (p < 0.05). This phenomenon may be related to the content of organic acids in the extract. However, the pH values of the tamarind extract loaded formulations were considered in the range of skin pH values (Ono et al., 2015).

Viscosity and Rheological Measurement
The flow characteristic comparison of three formulations are displayed in Figure 2.
According to Figure 2, in general, the viscosity of the lotions tended to decrease when the shear rates increased. The prepared lotions appeared to exhibit a shear-thinning system (Non-Newtonian flow). The viscosity of the formulations decreased when the shear rates increased. At speeds of 45, 60 and 90 rpm, there was no statistical difference in the viscosity between F0 and F1 (p> 0.05).
However, the viscosity of F2 was significantly lower than that of the other two formulations (p< 0.05). The decreased viscosity of F2, which contained 4%w/w tamarind extract, was probably caused by high content of organic acids in the extract as reported by Garcia-Olvera, Reilly, Lehmann, and Alvarado (2016). The viscosity values of the tamarind fruit pulp extract loaded formulations were rather low < 1,000 cps. Therefore, they are expected to be easily applied on the skin surface.

Antioxidant Activity of Tamarind Fruit Pulp Extract Loaded Lotions
For the lotions formulations, the radical scavening activity was expressed as percetage of reduction of initial radical absorbance by formulations at diffrerent concentrations ( Figure 3).
Ascorbic acid was used as a positive control with EC50 of 2.795 µg/mL. The unloaded formulation F0 did not have any antioxidant activity based on %SCV activity values at all concentrations tested.
In contrast, both tamarind extract loaded formulations F1 and F2 displayed the existence of antioxidant activity as shown in Figure 4. According to these results, it can be speculated that the antioxidant properties were solely caused by tamarind fruit pulp extract which was incorporated in the lotion bases. It was found that the formulation F2 had markedly higher %SCV compared to the formulation F1. This phenomenon may be related to high content of tamarind fruit pulp extract in the formulation that can give more active ingredient(s) to inhibit the oxidation process.

Conclusion
The current results demonstrated that tamarind fruit pulp extract was successfully

Scope of Future Research
In the future, the stability should be performed for short and long term to know the storage time of the product.

Research Limitation
In this research, we formulated tamarind fruit pulp extract in lotion which had large droplet size. Large droplet size will be affected to penetration of active compound through the skin. The limitation data of droplet size was the major problem to know the formulation can be easly penetrate through the skin or not.