A Review On Polyherbal Formulations For Immune Enhancement: Phytochemical Standardization, Antioxidant Potential, And Immunomodulatory Activity Of Traditional Rasayana Herbs

Immunity is the most important system of the body. It is categorized as first-line, non-specific, and at times-specific response to pathogens to protect the body from any antigenic insult without creating damage to host tissue[1,6]. There are two types of immunity: innate/ non-specific immunity which involves the action of macrophages, neutrophils, natural killer cells, and complement system; and adaptive/ acquried/specific immunity with involves B-lymphocytes, T-lymphocytes, memory cells, and immunoglobulins [9].

Recent crisis of global disease burden has given unforgettable pace to the need for preventive immune-stimulant in human life. The burden of emerging and re-emerging viral diseases, chronic stress, erratic lifestyles, nutritional deficiency, and the development of antibiotic resistance in microbes has drastically deteriorated the immune potential in recent times [3,14]. This demands the maintenance of innate immunity rather than treatment after the establishment of infection in humans. Ayurveda mentions herbs for the improvement of the immune system under the category of Rasayana drugs.

Rasayana drugs are stated to have tissue promotive action which complete nourishment and responsible for longevity, improved memory, retentions, intelligence, youthfulness, preeminence in complexion and luster, optimum development of physique and senseorgans, mastery over speech, respectability and brilliance [12]. In the classical Sanskrit literature, the word Rasayana has been described as a means of functional nourishment and one obtaining such a state is not affected by any disease. Pharmacological evaluation of Rasayana drugs has proved many of these claims [12,19,28]. Herbal drug development in the form of proper dosage formulation that can be standardized is a challenging task. Since the raw material for plant -based medicines is derived from a wide variety of geographical sources, standardization of finished medicine or the product is of utmost importance. Dispersible tablets are an attractive dosage form for developing and marketing polyherbal immunobooster formulations since they are easy to administer in pediatric and geriatric patients and do not require water for dissolution so dissolution takes place quickly in the neutral condition of the oral cavity [13,16].

In this review, the development of dispersible polyherbal immunobooster tablet- phytochemical standardization, antioxidant, immunomodulatory evaluation, and stability study evidence has been summarized for a total of 6 Rasayana botanicals.

BOTANICAL PROFILE OF IMPORTANT IMMUNOMODULATORY HERBS

1. Emblica officinalis (Amla)

Amla is the richest natural source of vitamin C [8,11]. Amla has reportedly 20 times higher vitamin C content than citrus fruits, in addition to emblicanina-A, pedunculagin, punigluconin and emblicanin-B. It is considered to be the best among

rejuvenative herbs in Ayurveda, and is useful as vayasthapana (arresting age), medhya (general tonic and memory enhancer) and for immunomodulation. Emblica officinalis enhances natural killer cell activity and phagocytosis by macrophages as well as haematopoiesis, as evidenced by increased IgM and IgG titer in in vivo studies. It is also found to be hepatoprotectant.[4,5]

2. Tinospora cordifolia (Giloy / Guduchi)

Giloy is highly regarded by present in immunologist as it is well proven to act on the innate immune system (Nikhil Sharma, YogendraKrishna MS et al. studied the immunomodulatory activity of Guduchi in patients with Dengue and allergic rhinitis. The results suggest improvement in platelet count and the neutrophil to lymphocyte ratio. Stress is widely considered to be immunosuppressive. Giloy is adaptogen and immunostimulating to protect immune balance in the body during a stressful situation. During infection, this activity is beneficial in promoting recovery while in normal health it may provide resistance to system stress. Both these roles support its recognition as a Tridoshanashak Rasayana dravya in Ayurveda classical text. [15,19]

3. Withania somnifera (Ashwagandha)

Over thousands of years (and more so in the past two decades of exhaustive research), Ashwagandha has been excessively explored for its immunopotentiator, adaptogenic, antioxidant, antistressor, and anabolic effect. It is rejuvenator in classical Ayurvedic text and promising clinical studies are available on its immunomodulatory and stress-mitigating activity. In clinical trials on elderly ill-appearing persons, Ashwagandha supplementation has led to a significant increase in IgG subclasses, especially IgG2 levels, and overall immunity perks up.[12,

Withanolides are the steroidal alkalids responsible for medicinal activity in Ashwagandha [1,8]. In clinical trials on adults under extreme psychological stress and consequent low level of immunity, Ashwagandha supplementation has led to an augmented T cell count (notably an increased CD4+ T cell count) and increased and increased serumit is through the suppression of the master regulator of inflammation, NF-κB, that and increased serum immunoglobulin [2,10].

4. Ocimum sanctum (Tulsi)

Tulsi has been regarded as universal protector of health in Indian tradition. It is marginally immunomodulator in action and enriches the phytochemical profile of immunostimulatory polyherbal formulation with its eugenol, and antioxidants.

In clinical trial on healthy persons, Tulsi has promoted antibody production, macrophage migration index and IFN-gamma production.[3,18]

The abundant flavonoid apigenin in Tulsi is responsible for its measurable suppression of inflammation through a marked inhibition of pro-inflammatory prostaglandin E2 via down-regulation of the cox-2 enzyme, without impeding physiological levels of inflammation required for protection against infection as evidenced by amelioration of clinical symptoms of fever induction and systemic-inflammation (“overall symptomatic score”) despite simultaneous increase in IFN-gamma and rise of body temperature. [31] 2.5.Curcuma longa (Haldi) and Zingiber officinale (Ginger)

Perhaps the most famous popular and most studied phytochemical ever, the bright yellow pigment and key pharmacophore of turmeric, curcumin, is aniallenged anti-inflammatory and anti-oxidant agent [13,25]. In support of the extensive body of evidences for these actions, curcumin is also able to scatter cytokine production and messa regulated inflammation by immune cells by inhibiting master transcription fac activation of anti-bacterial peptides in the immune response, while at the same regulating protein that orchestrates excessive tissue-destructive inflammation. It is proven to stimulate T-cell proliferation and macrophage activation PTurmeric is always described as a critical immunomodulation in classical Ayurveda-literature.

Ginger compliments turmeric with its gingerol and shogaol content, responsible for antioand antimicrobial activity. In pre-clinical studies, ginger extracts have been shown to prolifenate T-c stimulate macrophages. TNF-alpha is a target in limiting the destructive potential of excessive inflammation in diseases like arthritis, and ginger has been mapped to modulate tissue destruction through this pathway in preclinical studies.[20,24]

RATIONALE FOR POLYHERBAL COMBINATION

The ancient Ayurvedic system of pharmacy puts forth the concept of Anupana, which means that the action of each herb is potentiated when administered in proper combination with others. Pharmacologically, such an effect is termed as synergism, where two or more phytochemically diverse herbs, acting through different molecular mechanisms but on the same immunological pathway, potentiate the effect that would be achieved if the individual ingredients were administered alone [9,16] In case of immunity, a polyherbal combination confers several apparent benefits. Emblica officinalis forms the core antioxidant scaffold and provides humoral immunity. Tinospora cordifolia and Withania somnifera provide direct macrophage activation and lymphocyte proliferation. Ocimum sanctum extends antiviral and anti-inflammatory support. Curcuma longa and Zingiber officinale counter oxidative burden and cytokine imbalance [6,13] Thus, the entire range of immune response, from barrier immunity to memory cells, is addressed using a lower dose of each ingredient, minimizing the risk of dose-dependent side effects. In addition, the polyherbal combination promotes mutually increased bioavailability. Piperine like alkaloids present in some of the above herbs inhibit p-Glycoprotein mediated efflux, therefore enhancing the mean residence time of curcumin, withanolides and other poorly available components in the intestinal mucosal cells.

This pharmacokinetic synergism is an important practical consideration in formulation design and helps explain why many traditional polyherbal preparations demonstrate clinical efficacy at doses where isolated phytochemicals would be pharmacologically inert [2,9].

FORMULATION DESIGN AND PHARMACEUTICAL DEVELOPMENT

1. Selection of Dosage Form

Dispersible tablets offer several important advantages over decoctions, powders and churna dosage forms for herbal immunoboosters. Dosing is accurate, shelf-life is prolonged due to minimal moisture interaction, and the main disadvantage of storage-induced oxidation of thermolabile phytoconstituents is eliminated [17,26] Dispersible tablets rapidly disintegrate in a small volume of water, usually within three minutes, to form a consistent suspension that is easier to swallow for geriatric and pediatric patients who are particularly at risk of immune suppression. If, after sieving herbal extracts through mesh #40, flow properties are sufficient, direct compression is the preferred method for manufacturing as it precludes the use of heat and moisture during wet granulation, which can destroy heat labile phenolics, flavonoids and essential oil components that contribute strongly to immunopharmacological activity [20,24].

2. Excipient Selection and Rationale

The use of crospovidone as the main super-disintegrant is justified by its more rapid water-uptake compared to croscarmellose sodium in the presence of tannins, which tend to interact with cellulosics and slow down water absorption [2,9,13]. 4.3 Approach to Formulation Optimization

Several trial batches were manufactured with variations in composition of superdisintegrants (5-8%), magnesium stearate (0.5-1.5%) and herbal extract to diluents ratio. The optimized batch was selected based on hardness, friability, disintegration time, wetting time, water absorption ratio and palatability. Average tablet weight is 400-500 mg and disintegration time of the optimized formulation is not more than three minutes in 200 mL water at room temperature.

EVALUATION PARAMETERS AND RESULTS

1. Physicochemical and Organoleptic Evaluation

Preliminary physicochemical testing of the formulation blend gave the following results, which are as expected for tannin and phenolic rich extracts.

Organoleptic evaluation revealed a light to dark brown appearance, aromatic odor mainly due to Amla and Ashwagandha components, and a bitterastringent aftertaste which was reduced by the added sweetener and flavor. [5,17] Physicochemical data are displayed in the table below:

Low moisture content (2.78%) is an important indication of manufacturability and storability of the formulation, since excessive moisture breeds growth of microbes as well resulting in hydrolysis of glycosidic bonds and esters that are phytochemically rampant in the formulation ingredients [28,29].

2. Phytochemical Screening

Phytochemical screening of the combined extract revealed the presence of all major classes of immunologically active secondary metabolites. Dragendorff and Mayer tests were positive for alkaloids, the Shinoda test indicated the presence of flavonoids, Ferric Chloride reaction indicated the presence of both tannins and phenolic compounds, Froth test confirmed the presence of saponins and the Keller-Killiani test was positive for cardiac glycosides. Results are depicted in the following table:

The confirmed presence of flavonoids and phenolic compounds is especially significant, since these compounds regulate immunity by scavenging reactive oxygen species that inhibit lymphocyte proliferation, by down regulating pro-inflammatory mediators and by initiating a cascade of intracellular immune-signaling events [11,19]. Saponins have been independently indicated in enhancing phagocytic activity of macrophages and stimulating mucosal immunity.

3. Powder Flow and Compressibility

Flow properties within pharmacopoeially acceptable limits indicate that the blend can be compressed directly without the risk of dose variation. A compressibility index (Carr's index) below 15% and degree of flowability (Hausner ratio) below 1.25 are indicators of favorable balance between inter-particle cohesive and repulsive forces [18] and these observations jointly validate the suitability of direct compression approach to formulation manufacture.

4. Antioxidant Activity: DPPH Free Radical Scavenging

The DPPH (1,1-diphenyl-2-picrylhydrazyl) method is the most commonly cited procedure for determining anti-oxidant activity of herbal extracts, and may be considered as an indicator of immune protection. The following results were obtained for the formulation under investigation

The value is comparable to data reported for other polyherbal formulations with immunomodulatory activity. There is a considerable difference in activity between the formulation and the vitamin C reference standard, but this is due to curcuminoids from Curcuma longa and ascorbic acid equivalents from Emblica officinalis are responsible for the majority of observed anti-oxidant activity [4,13,23]

Immunologically speaking, high anti-oxidant activity prevents oxidative damage to cellular and organelle lipid membranes of immune cells, fragmentation of lymphocyte DNA and free radicalinduced cytokine imbalance which is the main cause for reduced acquired immunity in chronic metabolic disorders and cancers [5,11]

5. Immunomodulatory Activity: Carbon Clearance Assay

The carbon clearance assay provides a measure of phagocytic activity of the extract on in vitro incubation with murine peritoneal macrophages. Increase in phagocytic index compared to control is indicative of enhanced macrophage activation and acquired immunity. The following results were obtained: