Aloe vera L. Gel: Biochemical Composition ... - MAFIADOC.COM

1 Aloe vera L. Gel: Biochemical Composition, Processing and Nutraceutical Applications ANIRBAN RAY1* AND SAMPAD GHOSH2

ABSTRACT

The nutraceutical and cosmeceutical potential of Aloe vera L. (family: Xanthorrhoeaceae) in a myriad of varied responses has been turned out for human well-being over the years. Polysaccharides are demonstrated as the major component compounds for the bioactivities of A. vera gel. Irrespective of the polysaccharides, the synergism among more than 200 biologically active components of aloe gel cannot be ruled out to demonstrate the pertained bioactivities. The composition and proportion of gel-components significantly influence the bioactive potential of aloe gel. The successive value addition in one or more components of the value chain of aloe gel processing depends on the client-mediated identification of the most critical missing links and bridges them through research-driven interventions. In this respect, a succinct resume of information pertained to the biochemical composition, processing and biological application of aloe gel would be of help in optimizing the component of the value chain of aloe processing. Key words:

Aloe vera L., Aloe gel composition, Processing, Bioactivity

Agricultural and Food Engineering Department, Indian Institute of Technology, Kharagpur, West Bengal, Kharagpur 721302, India 2 Department of Chemistry, National Institute of Technology, Jamshedpur, Jharkhand 831014, India * Corresponding author: E-mail: [email protected] 1

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1. INTRODUCTION

Aloe vera L. (syn. Aloe barbadensis Miller), a miraculous plant, has a long history as a multipurpose folk remedy. Its name is most likely derived from the Arabic word Alloeh, meaning “shining bitter substance”. Aloe vera L. (A. vera) belongs to the family Xanthorrhoeaceae and sub-family Asphodeloideae (Angiosperm Phylogeny Group III System, 2009)[1], is the most familiar and popular medicinal plant among the other species of Aloe genus. Tropical Africa is the original habitat of Aloe species but now A. vera farming is gaining up momentum in warm climate areas of Asia, Europe and America on account of its growing importance and increasing market demand. There are two beneficial products extracted from aloe leaves, namely ‘gel’ and ‘sap’, both of which show potential bio-activity with reference to pharmacological applications[2–4]. Colorless astringent ‘aloe gel’ (AG) composed of parenchyma tissues is one of the widely used natural products in the world having unique immune boosting potential. Many biological activities including anti-viral, anti-bacterial, laxative, protection against radiation, anti-oxidant, antiinflammation, anti-cancer, anti-diabetic, anti-allergic and immunostimulation have been attributed to ‘aloe gel’[2–6]. In this context, the present article describes a succinct resume of information pertained to biochemical composition, processing and application of A. vera gel. 2. PHYTOGRAPHY, ECONOMIC IMPORTANCE AND DEFINITION OF ALOE VERA GEL

Aloe vera L. (syn. A. barbadensis Miller) is a traditionally used medicinal plant which was regarded as the ‘universal panacea’ by Greek scientists 2000 years ago. The Egyptians called aloe ‘the plant of immortality’, and the American Indians called aloe ‘the wand of heaven’. Generally, the plant height ranges from 0.60 m to 1.50 m, and the leaves can be spread up to 60 cm in length and leaves have thick epidermis or skin covered with cuticle (Fig. 1a). Turgid green leaves, having 40–50 cm × 6–7 cm in size, are present in dense rosette pattern. Leaf lamina is acuminated in shape, and leaf margin are serrated with firm spines (> 2 mm). Leaf blade is concave in upper surface, and convex at the lower surface. Leaves have thick epidermis or skin covered with cuticle, commonly known as outer rind. Inside the thick epidermis, highly viscous, transparent and odorless parenchyma is present. Aloe gel is a clear translucent mucilaginous parenchyma which is obtained by removing the epidermal part. The plants are propagated vegetatively by suckers which are produced at the base of the plants. The inflorescences are terminal and dense raceme in structure. Aloe is referred to as a “healing plant”, and is a source of two main bioactive products. The first of these two products is the yellowish ‘exudate’ from the cut leaf base and contains a high amount of anthraquinone compounds such as aloin, aloe-emodin and emodin. The astringent exudate

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can be used as potent antioxidant, cathartic and lacquer agents[5]. The second product is mucilaginous ‘aloe gel’, having a diverse array of pharmacological attributes[5,6]. Many biological activities including anti-viral, anti-bacterial, laxative, protection against radiation, anti-oxidant, anti-inflammation, anticancer, anti-diabetic, anti-allergic and immuno-stimulation have been attributed to this parenchymatous gel [2–9] . A large part of these pharmacological properties accounts for the presence of various bioactive polysaccharides, which constitute the major fraction of AG[1,6,9–12]. AG has also wound and burn healing properties with potential humectant activity[4,7,12,13]. It has been suggested that the physical, biochemical and biological activities of AG are the results of the synergism among the components rather than a single chemical substance[6,14], and variations in the structural components of gel may alter its potential usage. Other than the pharmacological properties, Aloe is also used as one of functional ingredients in food to fortify the nutritive values of the food products[6]. In the food industry, it has been used as a source of functional foods and as an ingredient in other food products, for the production of health drinks and beverages[9]. AG has also been used as base material for the production of creams, lotions, soaps, shampoos, facial cleansers and other products in the cosmetic and toiletry industry[4,7,14]. The ambiguity on account of the nomenclature of ‘AG’ has resulted in different terminologies that are assigned to describe the gel. The core part of the aloe leaf composed of parenchyma and designated with several names such as inner pulp, mucilage tissue, mucilaginous gel, mucilaginous jelly, inner gel and so on[9,10,15]. Inner pulps of aloe leaves are composed of cell walls, degenerated cellular organelles, mucilaginous gel and water. AG refers to the translucent, colloidal mannose rich fraction of parenchyma tissue and consists of about 99.50% water[1,9,14]. Generally, parenchymatous inner pulp of aloe leaves is considered to be ‘aloe gel’. The cross section of A. vera leaf and the presence of AG matrix are shown in Fig. 1b.

Fig. 1: (a) Aloe vera L. plant, and (b) cross section of aloe leaf (45 ×)

It is worthy to mention that plant cell wall, mainly composed of cellulosic fibers, consists of macro and micro-fibrils, which are composed of polymeric

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chain of cellulose molecules (Fig. 2). Successive cellulosic molecules are joined by primary valencies to get a chainlike arrangement and laterally held by hydrogen bonds, leading to a cellulosic micelle-matrix. Several integral molecules (pectin, hemicelluloses, protein and lipids) existing in the void space of the cellulosic fibers facilitate the inter-cellulosic fiber cross linkage and form a dense matrix, mainly composed of polysaccharides[16,17]. The water holding and swelling mechanism of plant cell wall matrix (here parenchyma cell wall of AG) implicitly dependent on the attraction and repulsion forces exist in the diffuse double layer of the fibrillar surfaces, considering the degree of inter-fibrillar branching. According to Crasnier et al.[18] plant cell wall acts as polyanionic matrix, and considered as ion exchangers in relation to the cellulosic cell-wall matrix ultra-structure and associated inter-fibrillar branching, electrical properties; and the presence of different minerals have significance in the regulation of cell wall swelling capacity of plant cells[4,19,20].

Fig. 2: Simplified illustration of plant cell wall ultra-structure

3. BIOCHEMICAL COMPOSITION AND ITS POTENTIAL IMPLICATION WITH BIOACTIVITY, AND QUALITY CONTROL OF THE GEL

3.1. Biochemical Characterization of AG The biological activity of any substance is mainly implicated by its compositional features. So, it is necessary to understand the biochemical properties and the proportions of the active principals contained therein in regard to evaluate the bio-active potential to ensure the effective application of AG. The biochemical properties of AG have been studied by different group of researchers. AG mainly composed of mannose, glucose and galactose monomers which constitute the structural reserve polysaccharide acemannan or -(1, 4)-linked acetylated mannose (Fig. 3), a functional carbohydrate in AG[1,11]. The structural monsaccharides are present in (1,4)-

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linked mannoses, 1 (1,4)-linked glucose, and 1 (1-6)-linked galactose form, and some of the mannose monomers are modified by addition of an acetyl group.

Fig. 3: Basic molecular Structure of acetylated mannan or acemannan, a predominant component polysaccharide of Aloe vera gel. Inset: structure of acetyl group

The molar ratio of sugar and linked acetyl group present in this molecule is 1:3[21]. The polysaccharides contain 14 glycosidic linkages in between glucose and mannose moiety (glucose: mannose = 1:2.8). Aldopentose, acetylated glucomannan, galactogalacturan, glucogalactomannan, galactoglucoarabinomannan are also the important oligosaccharides or polysaccharides present in AG [6,9]. The average molecular weight of polysaccharides of the gel was found to be 2 million Da or above and constitutes approximately 0.20% – 0.30% of fresh AG[22]. Some time the polysaccharides in AG are attached with protein moiety and form glycoprotein molecules. Lectin and alprogen are the predominant glycolproteins found to be present in AG[23,24]. The polysaccharide molecules can hold water molecules in inter-molecular space and attributes the humectant activity to AG. These molecules also modulate the thermo stability and gelatinous aspect of the gel. Bioactivity of AG in relation to the conformation and the configuration of the component polysaccharides were studied by Leung et al.[25]. Three purified polysaccharide fractions (assigned as PAC-I, PAC-II and PAC-III) were separated from A. vera var. chinensis (Haw.) by membrane fractionation and gel filtration HPLC. The molecular weights of the fractionated polysaccharides were 10,000 kDa, 1300 kDa, and 470 kDa, respectively. The main sugar residue had been identified in the polysaccharide fractions was mannose. The quantity of mannose was found to be 91.50% in PAC-I, 87.9% in PAC-II and 53.7% in PAC-III. 1H NMR stereoscopy proved the structural similarities of polysaccharides present in PAC-I and PAC-II. The main configuration of PAC-I and PAC-II is (14)-D linked mannose with acetylation at C-6 of manopyranosyl unit. The observation was also supported by Kostalova et al.[26]. The structural polysaccharides are considered to be the main fraction of AG attributes the

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immunomodulatory and functional properties to A. vera. Different preclinical (in animals) and clinical (in humans) experiments confirmed the potency of the gel to act as biological response modifier, but in some cases the desired biological potency was not evinced. These discrepancies may be due to the dissimilarities in the source of the gel, cultivation procedures or post harvest gel processing. Apart from the polysaccharides, phenolic compounds e.g., flavonoids, flavonols, anthraquinones are also present in AG[2,3,27"29]. Although, the phenolics are the minority compounds in the gel[6], some of the biological activities viz. antioxidant activity, cathartic activity, UV opacity are implicated by the component phenolics. The phenolic contents are not inherent to aloe parenchyma. Aloe specific phenolics are mainly synthesized in the pericyclic spaces and readily diffuse to the parenchyma tissue on standing[30]. Generally, phenolic content has found to be low in field grown plants as compared to micro-propagated or in vitro plants[29]. The yield of different extraction techniques (viz. shaking, reflux, decoction etc.) and various extraction solvents such as absolute ethanol, methanol, aqueous ethanol and aqueous methanol on total phenol content were investigated by Sultana et al.[31]. A decrease in the content of phenolics was observed with the extracts prepared by reflux method. Among the solvents, higher extract yield and phenol content were obtained with aqueous organic solvents, especially with 80% (v/v) methanolic extraction techniques. Flavonols such as myricetin, quercetin and kaempferol have been isolated from A. v era leaves by the same r esear cher s. Separation and characterization of the phenolic anthraquinones from AG, leaf skin and sap was conducted by Rajendran et al.[32]. FTIR, UV-Vis spectroscopy and fluorescence spectroscopy were employed in the characterization of aloe specific phenolic anthraquinones. Aloin, aloe-emodin, aloetic acid, anthranol, emodin, ester of cinnamic acid and others are the main fraction of phenolic anthraquinones of AG. Aloin is an anthraquinones-glycoside composed of an anthraquinone and sugar moiety, and it is a mixture of two dia-stereoisomeric forms, namely aloin A (barbaloin, 10S) and B (isobarbaloin, 10R)[33"35]. Aloin is described as the phenolic anthraquinone maker of AG ascribing pro-oxidant effect, DNA-breakage preventing potential and cathartic activity to the gel[2,3,36]. The structure of aloin-A and B is shown in Fig. 4. Low molecular weight substances such as aloesin (chromone derivatives isolated from A. vera), -sitosterol, di-ethyl-hexyl-phthalate, arachidonic acid, cholesterol, gibberellin, lectin-like substance, lignins, salicylic acid, triglycerides, uronic acid etc. are also present in AG[6]. Aloesin, [2-acetonyl8-beta-d-glucopyranosyl-7-hydroxy-5-methylchromone], a natural hydroxyl methyl chromone compound isolated from the Aloe plants, is shown to have anti-melanogenesis potential via competitive inhibition of tyrosinase[37] and an anti-inflammatory activity as well[38]. The quantification of lignin and

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Fig. 4: General structure of Aloin, a member of anthracene glycosides. It may exist in two diastereoisomeric forms, namely Aloin A and Aloin B. Aloin A contains ‘H’ at R1 and Glucosyl at R2, for Aloin B the configuration is vice versa

uronic acid in AG was conducted by Femenia et al.[11,12]. The different enzymes present in gel are amylase, alkaline phosphatase, carboxypeptidase, catalase, cyclooxydase, lipase, Superoxide-dismutase etc. Catalase and superoxide-dismutase are the bio-active enzymes which synergies the antioxidant activity of AG. The presence of bradykinase, cellulase, carboxypeptidase, catalase, amylase and oxidase in AG was also observed[6,9]. Retinol (vitamin A), thiamine (vitamin B1), riboflavin (vitamin B2), niacin (vitamin B3), folic acid (vitamin B9) ascorbic acid (vitamin C) and -tocopherol (vitamin E) are the predominant vitamins found to be present in aloe parenchyma, and increase the antioxidant potential of A. vera. Choline and are also present in AG[39,40]. It has been reported that there was also a trace amount of cyanocobalamin (vitamin B12) present in gel[40]. The presence of different minerals was also noticed in AG. Mineral elements maintain the structural integrity of the living tissues, maintain the liquidity of the body fluids, cofactor of several functional enzymes and collectively maintain the optimum functionality of the living cells[4,41]. AG is supplemented with different health drinks as a source of various macro and micro-nutrients[9,42]. Sodium and potassium are detected in higher amount as compared to calcium and Magnesium in AG, and presence of iron, copper, zinc and phosphorus was also detected in trace amount[11]. Similar observation was also made by Miranda et al.[42]. Quantity of calcium, magnesium and phosphorus at varying level was detected by Pandhair et al. [29] in both field gr own and micropropag ated Aloe plan ts [29] . Micropropagated aloes contain higher level of calcium content than that of the field grown AG. Whereas, a little fluctuation was noticed in phosphorus and magnesium contents among the micropropagated and field grown AG. According to Pandhair et al.[29], indole butyric acid treatment facilitated better root development and subsequent nutrient uptake results in higher degree of mineral accumulation in the micropropagated plants as compared to the conventionally field grown aloe. Rajendran et al.[43] reported that the concentration of K, Mg and Na in the gel was predominant over the other metal-ions. Recently, Ca, K, Mg, P, Na, Mn, Se, Al, Fe, Zn,Cu and Cd are

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detected in AG by atomic absorption spectroscopy and SEM-EDX analysis as well[1,4]. The contents of different minerals of AG change as a function of age of the plants and climatic seasons; the contents of Na, Ca, K, Mg, P and Cu are observed to be high in 2-year-old plants than that of the 3 and 4year-old plants. Moreover, the presence of Se, an anti-oxidative element, has been detected in AG, which is the first report of its finding[4]. The SEMEDX profile of freeze-dried AG is shown in Fig. 5 along with the detected minerals. The knowledge of mineral distribution in Aloe parenchyma at different harvesting regimens can be utilized in optimizing the component of the value chain on A. vera processing with reference to the pertained usage of the foliar AG as the functional component in food and in cosmeceutical applications with desired level of necessary minerals. Gong et al.[44] proposed that the formation of arbuscular mycorrhizal association with Aloe roots facilitate the enhanced mineral uptake from soil and make AG rich in various types of mineral components such as Ca, Cl, Cr, Cu, Fe, Mg, Mn, K, P, Na, Zn and so on. Mineral absorption of A. vera was also supported by Sharma et al.[45], and cultivation of Aloe is suggested as a promising option for waste and polluted water treatment. The arbuscular mycorrhizal association by A. vera can be utilized by the systematic cultivation of aloe in soil conservation for improved soil fertility[45,46].

Fig. 5: EDX spectrums of freeze-dried Aloe vera L. gel showing the presence of different minerals in aloe parenchyma

Physico-chemical properties of any biomaterials are implicitly regulated by the integral functional groups[1–3]. Detection of predominant functional groups was carried out by several investigators with the help of IR spectroscopy[1,2,32,47–49]. A representative ATR-FTIR profile of freeze-dried AG is shown in Fig. 6 showing the presence of phenolic –OH (corresponds to 3420.36 cm-1), –CH3 (corresponds to 2929.62cm–1), =CO (corresponds to

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1631.44 cm–1), COO– (corresponds to 1420.05 cm–1) and –COC (corresponds to 1077.24 cm–1) as the predominant functional groups.

Fig. 6: ATR-FTIR spectrum of freeze-dried Aloe vera L. gel

With reference to previous investigations, it can be concluded that AG mainly composed of six different types of compounds namely, poly and mono saccharides (mannose, glucose, glucomannan, acemannan etc.), phenolics, vitamins, enzymes, low molecular weight substances and minerals. 3.2. Freshness and Retrogradation of Aloe Gel, And Quality Assurance Processing which can cause a shift in molecular weight distribution of Aloe polysaccharides and other components, affects the gel quality and modulate the effective usage of AG[12,42,50–55]. 1H NMR spectroscopy has been described as a potential tool which can be opted to assure the quality or freshness of AG [1,49,56–58] . International Aloe Science Council (IASC) presently demonstrated NMR study as a fingerprinting tool for AG characterization, and a potential method for quality checking as well. The signal for acetyl group within 2.00–2.26 ppm, and characteristic proton shift of glucose (at around 5.13 ppm) and malic acid (at around 3.08 and 5.13 ppm) in 1H NMR spectrum corroborates the freshness of AG[1]. Fig. 7 shows representative 1 H NMR spectrum of freshly prepared freeze-dried AG. The proton shift at 2.05, 3.08 and 5.12 ppm, respectively suggests characteristic presence of acemannan, malic acid and glucose. The acetylation of A. vera specific polysaccharides is mainly associated with the presence of acetyated mannan or acemannan[47]. The presence or absence of organic acids in 1H NMR spectrum indicates the condition of foliar gel of aloe. The presence of malic acid, a native organic acid of AG an obvious product of Crassulacean acid metabolism, affirms the freshness of the gel. Whereas, the detection of

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non-indigenous organic acids, viz. lactic acid, acetic acid, succinic acid is quite undesirable and mostly associate with the microbial degradation, contamination, improper post harvest processing and the pertained retrogradation of the gel[1,57,58]. Apart from the presence of acetic acid in AG which is produced by the de-acetylation of acemannan by chemical degradation, detection of other undesirable organic acids is mainly associated with the microbial contamination. The presence of glucomannan and free glucose to mannose ratio greater than 2:1 are also considered to be an indicator of freshness of gel[59].

Fig. 7: 1H-NMR spectrum of freeze-dried Aloe vera L. gel

Qualitative and quantitative modifications in bioactive substances promote the variation in the biochemical properties of the AG[9]. The effect of different air drying temperature, as a cost-effective dehydration technique, on AG processing has also been studied and the dehydration at 60 to 70°C has been demonstrated as the ideal drying temperature for AG[9,12,51]. Miranda et al.[50] estimated polysaccharide and protein contents in AG dehydrated at various temperatures (50, 60, 70, 80 and 90°C), and it was observed that dehydration at 50 to 90°C significantly decreases the polysaccharide contents in AG, whereas protein contents remain unaffected at this temperature regimen. A similar kind of study was also carried out by Gulia et al.[51] and it was observed that the detected variations were found to be very less in ash, crude fibre, protein and fat contents under different drying temperature regimens; aloin content was decreased with the increasing dehydration temperature (50–80°C). Both group of the scientists[9,51] suggested that 70°C is the ideal temperature for air-drying where the physico-chemical properties of AG were conserved in a better way as compared to other processing temperatures.

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Apart from dehydration and processing, there are many factors that can influence the proportion of the structural and functional components of AG, which could explain the differences in the results observed in the literatures[6]. One of the important factors is the provenance (place of origin) or varieties of A. vera used in the investigations[60]. It has also been reported that other factors decisively influence the composition of AG, such as the seasons[1,3,4,49], level of light irradiance and exposure to light[1,61], the place of cultivation and cultivation methods[62"65]. Carbon allocation, soluble carbohydrate and aloin content were estimated in AG under different sunlight irradiance regimens by Paez et al.[61], and minor treatment effect due to the variations in sunlight irradiance on the concentration of carbohydrate and aloin in foliar gel was noted. Age of aloe plants has also found to play an important role in the biochemical properties of gel[1,27,49] and higher amount of polysaccharides and total flavonoid contents were detected in three-year-old aloe plants in summer as compared to two and four-year-plants from other seasons. It is worthy to mention that, the concentration of polysaccharides, minerals and phenolics of freshly harvested gel were measured from different age groups of aloe at different seasons by our research group[1,4,49]. Collectively, it has been observed that the age of the plants and the harvesting season make significant contribution toward the concentration of the component compounds. A high acemannan concentration was mainly associated with 3-year-old followed by four 4-year-old aloes in summer season. A. vera at various growth periods possesses minerals to different concentrations. The concentration of Na and Mg was high followed by Ca at different age groups, and higher accumulation of Mg, K and P was detected at two year old plants as compared to three and four-year-old plants. Presumably, the growth periods of the aloe plant plays a decisive factor by which the metabolic flux was modulated in such a way, the photosynthetic rate, carbon sequestration, carbon partitioning as well as the rate of utilization of organic acids for carbohydrate synthesis varies[1]. 4. GEL PROCESSING

The effective usage of the AG for various products formulation depends on the appropriate and optimized process methodologies. Intriguingly, there is no commercial preparation has been proved to be stable and assure complete conservation of the bioactivity of AG. Since, many of the constituent active principles appear to be deteriorated on storage, the use of fresh gel is recommended. The fresh gel is prepared by first harvesting the leaves followed by washing with water and may be with a mild chlorine solution. After that the outer layers of the leaves or epidermis are removed including the pericyclic cells, leaving a “fillet” of gel. Care should be taken not to tear the green rind which can contaminate the fillet with leaf exudate. The pasteurization at 75-80 °C for less than 3 min may be carried out to stabilize

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the gel by inactivating the enzymatic activities. In an average, one can expect a minimum 43% yield of aloe gel from a 1 kg aloe leaf. Aloe concentrates are prepared by taking the pure AG that has been filtered, decolorized, and without any of the adulterants. The vacuum distillation process may also be employed for removing the water content in gel. Generally, the solid fraction of AG constitutes only 0.5% of fresh foliar gel weight. It has already been discussed in the preceding section that the higher temperatures regimens held for a longer time may affect the chemical composition and significantly influence the bioactivity of the gel[51]. In the literatures there are different claims and counter-claims for the superiority of one or another process. It has been suggested by United Aloe Technologists association that heat during pasteurization is one of the main contributors of the stressors deteriorates the quality of gel; whereas high temperature short time processing with preferably with the application of a trivial amount of antioxidant such as ascorbic acid is a preferred option[66]. The structural integrity of mucopolysaccharides during storage was found to be conserved by the addition of other natural polysaccharides which act synergistically and ascribe thermal stability to the gel. The additives or the stabilizers must be chosen carefully when the gel is intended for pharmaceutical or cosmeceutical purposes where organoleptic properties are of major importance. A clear distinction should be maintained between the translucent parenchyma cells, the aloe gel and the bitter exudates from the cells associated with vascular bundles in the outer green rind of the leaf[67]. This distinction has sometimes been obscured by using the extracts of the whole leaf during preparation of the gel or allowing the exudates to infiltrate. The proposition of colorization and de-colorization of gels described above leads to confusion in ascribing bioactive potential to individual components. Since the prevailing synergism among the component compounds of AG potentiates the bioactivities of interest with reference to a myriad of nutraceutical and cosmeceutical applications, delicacy should be optimum during aloe processing. There may indeed be synergism, which would not appear if the fractions were kept separate. Considering the complexities inherent in aloe pharmacognosy might be better to be as rigorous as possible in separation, at least initially. The sequential steps for the whole leaf commercial gel processing are shown in Fig. 8. 4.1. Methods for Aloe Gel Processing The detailed methods and principles pertained to the various steps of commercial AG processing are adopted from Genemco Inc, are described below. Step 1: Initial cleaning of aloe leaves Most of the companies use a large pool filled with potable water for initial cleaning of aloe leaves. This could also be untreated ground water. The

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Initial cleaning of Aloe leaves  Sanitization of leaves  Rinsing  Removal of injured part of the leaves and hand separation of spines  Removal of the outer rind or epidermis from the inner filet of the leaves using filet machine or knives  Removal of the organic waste and/or yellow exudates  Grinding, homogenizing and sieving of the fillets  Enzymatic treatment of the gel extract  Removal of the insoluble fiber component  Activated charcoal mediated removal of phenolics  Plate and frame filtration  Pasteurization  Evaporation  Drying Fig. 8: Basic steps for the processing of Aloe vera L. gel

purpose for this tank is to soften and partially release the foreign particles and dirt from the outer surface of the leaves. The different procedures for initial cleaning are as follows: Immersion: The aloe leaves are allowed to immerse into the water with no further action and they could then be manually removed out of the vat or mechanically by conveyor. Agitation: Here, the tank has high pressure water jets creating a whirlpool in the soaking tank. The dirt and other foreign debris are removed more efficiently by this system. Brushing: The aloe leaves collected from soaking tank are picked to brush manually and again placed to the conveyer belt. This type of manual brushing is very gentle on the leaves, and removal of dirt and foreign material is optimal. This step is also may considered to be an inspection and/or rejection point. Higher labor cost is the downside of this step. After soaking and pre-cleaning the leaves are moved by a conveying technique which is consist of a conveyor belt, stainless steel mesh or polypropylene

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conveying system. Some of the companies use a fluid conveying system, where the aloe leaves are placed in a canal with water that is being pumped so that the flow moves the leaves along. Step 2: Sanitization of leaves Mechanical brushing is followed prior to the final washing and rinsing. The leaves are picked up mechanically from the conveyor and press them against two or more wiping with plastic brushes. The main purpose for this brushing is to remove dirt and other foreign materials from the epidermis of the leaves. One or several simultaneous lines of brushes could be installed considering the capacity of the system used. Generally the material of choice for these brushes is soft plastic, which is capable of removing unwanted material completely from the surface of leaves while being gentle enough to avoid external mechanical injury to the leaf. Step 3: Rinsing Rinsing is the final step for washing of the leaves. This could be considered to be the final cleaning to the leaves at the outset of the manufacturing processes. Different sanitizing agent viz. chlorine, quaternary ammonium salts and others are used for rinsing. Stainless steel, fiber glass or concrete are used to build the body of the rinsing. This process can also be a twostage cleansing method, first in a sanitizing solution and secondly with water to remove the sanitizing remains. Step 4: Removal of injured part of the leaves and hand separation of spines At this step, the leaves get inspected one by one as they enter the process according to the predetermined acceptance specifications. Leaves that do not meet the required attributes are rejected here. The leaves with mechanical injuries, cuts and lesions are summarily discarded to avoid contamination towards downstream processing of the gel. Layout for this station is usually a conveyor that feeds leaves coming from the rinse tank into the table. There is a conveyer system to carry waste out, and a series of tables or conveyors also to sway the leaves to the next processing step. The proximal, distal, and peripheral spines of the leaf are usually separated manually by using knives or any metal made sharp tool. Step 5: Removal of the outer rind or epidermis from the inner filet of the leaves using filet machine or knives The epidermis or outer rinds of the leaves are peeled off with motorized automated gel filleting machine. Sometimes hand filleting method has also been followed in lieu of automated gel filleting machine. The cost effectiveness is the main advantage of using a machine in terms of labor,

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but it is disadvantageous with reference to the yield of gel which may go down as compared to hand filleting. Step 6: Removal of the organic waste and yellow exudates All of the foliar epidermal layer and fibers are discarded at this stage. Sometimes, these organic wastes can be composted and recycled in the farm and being used as green manure. Step 7: Grinding, homogenizing and sieving of the fillets Irrespective of the whole leaf process or aloe fillet process (aloe leaf devoid of epidermis), a specially designed grinder is there to homogenize the pertained phyto-extract followed by sieving thru a mesh with a hole size about 5 mm in diameter. The most familiar grinder used in industry is a hammer mill, with non-swiveling hammers. This type of grinder is preferred for whole leaf aloe processing. The main purpose of this process is to extract the liquid gel contained therein. Usually, the peripheral and distal part is removed prior to grinding and the particle size is monitored changing the pore size of the mesh placed at the bottom of the grinder. Step 8: Enzymatic treatment of the gel extract In this step, newly extracted viscous AG is subjected to an enzymatic treatment to reduce the mucilaginous property in a manageable level. Liquid cellulase (approx. 20%, w/w) the most commonly used enzyme for enzymatic treatment of AG, is added to the vat, mixed and allowed to react for two to four hour of time at room temperature (25 ± 1ºC). Step 9: Removal of the insoluble fiber component The insoluble fiber resulting from crushing the whole leaf or the inner gel is removed by pulper. The device has a rotating portion that is either an endless screw or paddles that push the mixture against a screening or sieving apparatus. The liquid gel passes through the sieve, and the insoluble fiber components are pushed to the end of the machine. The pore size of the sieving apparatus may vary, ranged from 500–800 m. This machine allows to carry on coarse filtration very conveniently. Step 10: Activated charcoal mediated removal of phenolics Phenolics, particularly anthraquinones are considered to be the most significant undesirable components of AG. Although, aloin is a phenolic anthraquinone marker of AG attributes cathartic activity to aloe, the concentration of this phenoic compounds should be less in the processed gel. But in the case of whole leaf AG the presence of aloin and other phenolics is unavoidable. Activated charcoal is used to control or remove the phenolic contaminants, and seems to be very effective to mitigate this problem. In

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brief, the activated charcoal is added to a tank and then removed by filtration or by pumping the liquid through a column containing activated charcoal. Step 11: Plate and frame filtration A single filtration device or a series of filtration is carried out in this step to remove the charcoal added in the preceding step or to remove undesirable insoluble fiber components. Filtering aids can also be used to accomplish the filtration more efficiently. Most familiar type of filter used here is filter press; however other types of filters have also been used. Step 12: Pasteurization This is a thermal treatment to reduce the microbial contamination of the product. Typically, AG contains a natural flora of microorganisms, which are reproduced rapidly after the maceration of aloe parenchyma. So, at the downstream stages of the processing the microbial load needs to be reduced to assure the stability of the final product. The microbial load can be reduced by using a thermal treatment, referred to as pasteurization. There are mainly three types of pasteurization viz. Low temperature low time (LTLT), high temperature short time (HST) and ultra high temperature (UHT). LTLT: In this method the whole batch is placed in a tank and heated typically at 60 °C (140 °F) for 30 min. HTST: This is a continuous process where the product is heated very quickly for 30–60 s in a heat exchanger at 77°C (170°F), and the temperature is applied for a brief period of time (1 to 2 min). After heating, the product is allowed to cool at room temperature. HTST pasteurization uses a heat recovery system where the hot product is allowed to exchange heat with the incoming product. If the required final temperature of the product is lower than that of the room temperature an additional section of the heat exchanger is added; water from either a cooling tower or a process chiller is used for further cooling of the product. UHT: This strategy also considers a continuous process that works in a very similar fashion as the HTST, but the fluid which is used here is heated to higher temperature, (near to 110°C), though heat transfer is occurred very fast (2–5 s), and the peak of the temperature regimen is only held for a fraction of a second. Step13: Evaporation In this step the water activity of AG is reduced to increase and the self-life of gel powder for amenable transportation. The final products which are marketed in the liquid form range from five to forty times concentrated. Since, aloe is sensitive to heat the concentration must be maintained at the lowest possible temperature to attain decreased water activity. There

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are several designs of evaporators which work under high vacuum to allow water evaporation at low temperature range. It is also important to maximize the heat transfer efficacy to lower the operational cost of the unit, and therefore several evaporation chambers are placed in a series i.e., the hot evaporated gases go from the first to the second and so forth. These are referred to as multiple effect evaporators. Typical operating temperature is maintained at about 60 °C for this type of evaporation. Step14: Drying The marketable aloe powders are mostly in the powdered form; generally 200 × powder from aloe fillets or 100 × powders from whole leaf aloe are used to be marketed. Final evaporation must be performed at low temperature to preserve the natural components of aloe, and conserve the pertained synergisms among the components as well. This is especially important when the aloe solids become more concentrated, and hence they are more susceptible to react chemically between each other. Spray drying, one of the widely accepted drying methodology converts the aloe concentrate into a very fine mist of particles or droplets, is carried out in a conical shaped chamber where the concentrate encounters a flow of warm dry air. The underlying principle of the spray drying is to increases the surface to volume ratio of the AG droplets leading to increased evaporation of retained and adsorbed water molecules present therein. The droplets are suspended inside the chamber until they become more dense by the most possible evaporation and it is fallen to the bottom of the chamber in a powdered form and stored. There are two types of devices currently used to produce the fine mist of droplets. One is a very high pressure valve where the liquid is forced by 2000 psi of pressure (approx.) to go through a very small orifice, which allows the liquid to spray out in a very fine mist. The other consists of a rotating disk on which a stream of liquid is dropped. The centrifugal force of the spinning disk then jets out the liquid and converting it into a fine mist. Rotation of the disk used to be very fast and maintained at 10, 000 rpm (approx.). Air driven turbine is used to maintain the functionality of the disks properly. 5. BIOACTIVITIES OF ALOE VERA

A. vera seems to be the most promising candidate from aloe genus with diverse array of biological activities including anti-viral, anti-bacterial, laxative, protection against radiation, anti-oxidant, anti-inflammation, anticancer, anti-diabetic, anti-allergic, immuno-stimulation and UV protecting activity[1"5,49]. Different biological activities of Aloe vera gel are as follows:

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• Angiogenesis–  sitosterol is the main attributer of the formation of new blood vessels or angiogenesis. • Anti-ageing– Immunomodulation activity by acemannan and glucose6-phosphate moieties attributes anti-aging properties to aloe gel. • Anti-allergic– Several glycoprotein components ascribes to this activity. • Anti-cancer– Acemannan and phenolic anthraquinones of gel are considered to be prophylactic against certain types of cancer and many chronic degenerative diseases being the potential antioxidants or prooxidants with unique DNA breakage preventing potential. Acemannan prevent malignancy by its potential immune-stimulant activity. • Anti-diabetic– Aloe gel shows anti-diabetic properties by increasing blood insulin level and/or triggering the hepatic and extra-hepatic glucose utilization. • Anti-neoplastic activity – Acemannan, aloin, aloe-emodin, emodin, diethylhexylphthalate compounds are responsible compounds for antitumor activity. • Anti-obesity– Aloe gel shows anti-obesity activity by inhibiting pancreatic lipase, and leading to retarded fat metabolism and triacyl glycerol absorption as well. • Antioxidant– Mainly the phenolic anthraquinone compounds viz. aloin, aloe-emodin, aloeresin, aloenin etc. Presently acemannan has also been described as a unique antioxidant polysaccharide from aloe. • Anti-tyrosinase – Aloesin, salicylic acid are the main compound. Sunlight induced melanin synthesis leading to hyper-pigmentation can be prevented by inhibiting the tyrosinase enzyme (E.C. 1.14.18.1) activity. • Cathartic or laxative – Mainly the anthraquinones attribute cathartic or purgative activity to AG. Aloin, aloe-emodin and emodin are the major fraction of anthraquinones in aloe. • Dietary mineral source– It may be used as the dietary supplement of different minerals, viz. Ca, K, Mg, P, Na, Mn, Se, Al, Fe, Zn, Cu etc. • Moisturizer– Cutaneous application of aloe gel offers soothing moisturizing activity by its potential water retention capacity rendered by component polysaccharide compounds. • Wound healing– Acetylated mannan or acemannan and mannose-6phosphate are responsible for macrophage activation leading to wound healing and improved dermal health. 6. FUTURE PERSPECTIVES

Aloe vera is one of the major industrial plant and it supplies raw materials to pharmaceutical, food and cosmetic industries. It is among the few medicinal plants by virtue of their extensive medicinal, nutraceutical and

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other uses enjoy a major chunk of the market across the globe. Sometimes, it is not possible to exploit the excellent potential of this miraculous medicinal plant accounts for the lack of cultivation and processing knowhow leading to the yield of aloe based end products with little or virtually no active ingredients. Given the exponentially growing demand for aloe based products in the international market, A. vera presents the finest commercial opportunity among the various medicinal plants. With reference to the value chain of A. vera, this should be taken into account the synergies of research and development actors to cater the needs of small and marginal aloe farmers, and for efficient use of resources as well. Such, future research may aim to enhance gel quality for suitable value added aloe products, and develop more efficient process technology for production of A. vera gel fillet and powder. ACKNOWLEDGEMENTS

Indian Council of Agricultural Research–National Agricultural Innovation Project (ICAR–NAIP), Department of Science and Technology (DST), New Delhi, India and Agricultural and Food Engineering Department of Indian Institute of Technology (IIT), Kharagpur, India are acknowledged duly for this publication. REFERENCES [1] [2] [3] [4] [5] [6] [7]

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