FORMULATION AND EVALUATION OF HERBAL CAPSULE CONTAINING CURCUMA MANGGA VALETON and ZIJP. EXTRACT AND ITS IMMUNOSTIMULATORY EFFECTS

Objective : The present study was carried out to formulate and evaluate


INTRODUCTION
Multiple cells and molecules that can identify and eliminate different pathogens and undesirable substances are involved in human defense [1,2].Phagocytes are crucial to innate immunity, while lymphocytes primarily support adaptive immunity.T lymphocytes can be classified into two main lineages based on whether they express the CD4+ or CD8+proteins on their surface.Stimulation of CD4+T cell initiates delayed-type hypersensitivity (DTH) response [3].
Th-1, Th-2, Th-17, and CD4 T regulatory (Treg) cells are the four subpopulations of CD4 T cells that carry out the functions of CD4 T cells [4].B cells become plasma cells, which are capable of producing antibodies with the aid of th cells.A particular relationship between an antibody molecule (the Fab component) and an antigen allows each antibody to attach to only one antigen.Other antibody components communicate with phagocytes and other immune system components [5].Immunoglobulin A (IgA), IgD, IgE, IgG, and IgM are the five types of antibodies found in humans.IgG is the immunoglobulin that predominates among them in serum.IgM first appears in the blood during the initial response to a new antigen, followed by IgG.When the production of IgG rises, IgM production falls [6].
Pathological conditions can be driven on by immune system abnormalities or dysfunction.In order to manage and treat illnesses caused on by immune system defects or dysfunction, the immune response system must be modified [7].Immunomodulators are used to boost or reduce host defense responses in the treatment of disorders where compromised immune responses have a significant impact on the course of the disease.Immunostimulant medications are preferred for the treatment of immunodeficiency and infectious diseases, whereas immunosuppressive medications are frequently utilized to treat inflammation, allergy disorders, and organ rejection [8].
Since ancient times, the use of traditional medicines has increased.One of the medicinal plants utilized as alternative medicine is Curcuma mangga rhizome, which has been used to treat cancer, fever, and gastrointestinal disorders [9].Numerous active secondary metabolites, including flavonoids, saponins, glycosides, terpenoids, and steroids, were discovered to be present [10].Our prior studies showed that C. mangga has immunomodulatory effects on phagocytosis and antibody titer by in in vivo experiments [10,11].The toxic effect of C. mangga rhizomes was also assessed and it was shown that its LD50 was greater than 5000 mg/kg bw, indicating that it was not harmful [12].It has also been shown that the methanol extract of C. mangga exhibits nitric oxide inhibitory activity in vitro [13].The findings highlight C. mangga might be developed as an immunostimulatory agent.C. mangga extract need to be processed into a dosage form in order to produce a human immunostimulatory agent that is safe and efficient.
One of the most simple prepared dosage forms is capsule.This dosage form requires less method preparation compared to the other dosage form.The crucial factors that need to be considered in capsule preparation are the formulation and evaluation of the granule mass.The efficiency of capsule production, especially in hard capsule filling, is affected by the characteristics of granule mass, which related to the particle properties [14,15].The current study was performed to formulate the capsule containing dry extract of C. mangga and evaluate its immunostimulatory effects on humoral and cellular-mediated immunity.

Plant materials
The plant collection was carried out in North Sumatera, Indonesia.A scientist from Herbarium Medanense (MEDA), Universitas Sumatera Utara, Indonesia, recognized the plant sample with the identity number of 5669/MEDA/2021.

Extraction procedure
The rhizomes were dried, crushed, and then extracted using ethanol as the solvent by maceration.In brief, 500 g of the rhizomes of C. mangga were macerated in ethanol (1:10).The solvent was then eliminated with the help of a rotary evaporator to produce an ethanol extract of the C. mangga rhizomes [10].

Curcuma mangga dried extract (CMDE) preparation
There were two adsorbents used to prepare the C. mangga dried extract (CMDE), polyvinyl pyrolidon (PVP) and corn starch.The ratio between PVP and corn starch with C. mangga ethanol extract was 1:2 and 1:1.5, respectively.The C. mangga extract was mixed with each of the adsorbents and sieved to homogenize the size.Then the mixture was dried in the drying board.Since the flowing properties of extract-corn starch powder was not good, co-processing of the dried extract (extract with corn starch) in to a granule form was conducted.The co-process was done using 10% cassava starch mucilage with wet granulation method.The term CMDE-PVP and CMDE-CS were used to represent C. mangga dried extract with PVP as adsorbent and C. mangga dried extract with corn starch as adsorbent, respectively.
The dried extracts were evaluated for their LOD value and flowing properties, including flowing time, tapped density, bulk density, Carr's index, Hausner ratio and angle of repose [15].The flow ability was examined by measuring the length time for the bulk to flow from a funnel sing.The bulk density was determined by recording the volume (Vo) of a known weight (M) of the granules mixture and after taping until constant volume (Vt) in a measuring cylinder.The bulk density (Ρb) and tapped density (Ρt) were calculated as M/Vo and M/Vt, respectively.The Carr index (CI%) was determined from Pb and Pt as Cl = Pt−Pb

Pt
The Hausner ratio (HR) was determined using the formula of HR =

Pt Pb
The angle of repose (α) was determined by measuring the height (h) and the diameter (d) when a conical pie formed from flowing over a funnel and then calculated the tangent as follow: ; α was then deduced from its tangent [15].

Granule mass preparation
The dried extracts were mixed with the excipients to prepare the granule mass for the capsule.The formulation of the granule mass was differentiated into 6 formulas with 3 different fillers, namely lactose, Avicel® and maltodextrin.Capsule shell size number 3 was chosen to prepare the capsule.The mass for each capsule was determined by subtracting the total weight of a filled capsule with the capsule shell.The capsule capacity obtained was 170 mg.The formula of the granule mass for capsule preparation can be seen in table 1.

Granule mass evaluation
Each granule mass formula was evaluated in term of water content, flow ability, tapped density, bulk density, Carr's index, Hausner ratio, angle of repose according to the method described in Carbinatto et al. [15].

Curcuma mangga capsule preparation and evaluation
Capsules were prepared with semi-automatic capsule filler.Capsule formula that met requirement of Indonesian Pharmacopoeia and showed the best-flowing properties was chosen to perform the in vivo study.Prepared capsules were evaluated for their weight uniformity and disintegration time according to the procedure explained in Indonesian Pharmacopoeia.Weight uniformity was determined by calculating the weight % deviations of 20 capsules [16].

Antigen preparation
Nutrient broth agar (NB) was used to grow Staphylococcus aureus, which was then incubated for 24 h.A 1 ml aliquot was mixed with 9 ml of NB, and the cell concentration was determined using the spectrophotometry method until 1 x 10 8 cells/ml was reached.It was also centrifuged for 10 min at 250 °C at 10.000 rpm.The supernatant was discarded.Thereafter, 1 ml of PBS was used to resuspend the cells.

Delayed-type hypersensitivity (DTH) response
DTH response assay was performed to determine the effect of C. mangga capsule on cellular immunity.The experiment was carried out by measuring paw volume following a modified previous study [17].Animals were given capsules containing C. mangga extract (100, 200, or 400 mg/kg BW) orally 72 h before being exposed to S. aureus (1 x 10 8 cells/ml) by intraperitoneal injection.This treatment was continued for 14 d.The negative control group, meanwhile, received avicel as a component of the basic formula.A positive control was Stimuno® (50 mg/kg BW).On day 14, the paw volume was measured, then, all the rats were challenged by same amount of S. aureus in hind footpad.

Immunomodulatory assay on antibody production
A modified method of an earlier study was used to assess the impact of the extract on Immunoglobulin G (IgG) production [17].The animals were given capsule containing C. mangga extract at dosages of 100, 200, and 400 mg/kg bw 72 h before getting sensitized to S. aureus (1 x 10 8 cells/ml) by intraperitoneal injections.The treatment continued for 14 d.The negative control group received avicel as a component of the basic formula.A positive control was Stimuno® (50 mg/kg bw).After the experiment, blood was drawn, and the serum was collected to measure the IgG levels.

Statistical analysis
The data were presented as mean±SD and analyzed using Kruskal-Wallis and Post hoc Mann-Whitney.P<0.05 was indicated as a significant difference.

Characteristic of Curcuma mangga dried extract (CMDE)
The dried extract of C. mangga showed an orange-brown colour.The colour of the dried extract is different according to the type of plant used in extraction.This result was supported by a previous study which reported that the dried extract of Picria fel-terrae leaves was greenish brown [18].The part of the plant used in the extraction and the content of secondary metabolites in the plant part also interfered to the colour of the extract.The adsorbents used in the drying process of C. mangga ethanol extract were PVP and corn starch.These two adsorbents gave different characteristics of the dried extract.The extract that dried with PVP had granule form and the extract dried with corn starch had fine powder form with smaller particle size (fig.1).The extract that dried with corn starch (CMDE-CS) showed bad flowing property due to the very fine particle size.However, CMDE-CS had lower water content compared to the CMDE-PVP.Therefore, CMDE-CS was co-processed in to granule form to gain better-flowing property.The characteristics of CMDE-PVP and CMDE-CS are presented in table 2.

Characteristic of capsule granule mass
The addition of excipients in granule mass for capsule preparation was aimed to increase the properties of the dried extract and to fill the space in the capsule shell.The mixture of all of the ingredients was shown in fig. 2. Generally, each formula of granule mass gave shorter flowing time and higher Carr's index compared to the dried extract alone.Particularly, in the granule mass formula using CMDE-CS showed slightly higher bulk and tapped densities compared with the CMDE-CS alone.However, formula with CMDE-PVP demonstrated a significant increase of bulk and tapped densities value compared to the CMDE-PVP alone.The influence of fillers addition also exhibited differences between the formula.The most significant effect of the fillers was noticed in the Carr's index parameter.The smallest Carr's index was generated by maltodextrin as seen in F3 (with CMDE-CS) and F6 (with CMDE-PVP).The characteristic results of capsule granule mass can be seen in table 3.

Weight uniformity
Capsule weight uniformity was conducted to ensure that each capsule contained a consistent dose of CMDE.Indonesian Pharmacopoeia stated that in 20 capsules, there should not more than 2 capsules has a deviation more than 7.5% and none of the capsule exceed 15% deviation [16].As shown in table 4, F1 had one capsule with deviation value more than 15%; F3 had 7 capsules that exceed 7.5%; F4, F5 and F6 also showed capsules that did not meet the requirement of Indonesian Pharmacopoeia.There was only F2 capsule formula that had fulfilled the Indonesian Pharmacopoeia requirement.

Disintegration time
The disintegration time of formula prepared was 3.20±0.67,2.15±0.76;3.20±1.11;10.16±5.56,3.46±1.37,and 4.13±0.84for F1-F6, respectively.All of the formula met the requirement of Indonesian Pharmacopoeia disintegration time, which is not more than 15 min.The fastest formula to disintegrate was shown by F2 with 2.15±0.76min.According to the capsule evaluation, it was obtained that F2 met all the requirements of Indonesian Pharmacopoeia in terms of weight uniformity and disintegration time.This formula was then chosen as the formula to prepare capsule dosage form for animal studies.

Delayed-type hypersensitivity (DTH) response
The administration of C. mangga capsule of formula F2 enhanced the footpad thickness of rats as compared to those of negative control (P<0.05).Of all samples tested, C. mangga capsule at dose of 400 mg/kg bw demonstrated the highest stimulation on DTH response (fig.3).Suprisingly, the stimulation was higher than those of positive control with paw volume of 3.14±0.19;2.14±0.21,for C. mangga capsule and positive control, respectively.

DISCUSSION
The bulk densities of the formula containing CMDE-PVP was higher than 0.5 g/ml, indicating that C. mangga granules mass had denser characteristic as compared to the those of formula containing CMDE-CS.Usually denser particles experience great limitation to flow due to the particles contact to each other.The bulk density value for each formula was lower than the tapped density value, indicating that there were empty spaces between the particles.However, the difference between tapped and bulk densities of formula containing CMDE-PVP showed greater compared to the tapped and bulk densities difference of formula containing CMDE-CS.The difference between bulk and tapped densities is related to the inter-particle porosity.Greater the difference between tapped and bulk densities resulting higher value of inter-particle porosity.Hence, there are many of pores and empty space between the particles in the formula.
Short flowing time below than 10 seconds indicates that the formula has good flowability.All of the formula showed flowing time between 1.05±0.05 to 1.60±0.08seconds.It indicated that all of the formula had excellent flowing property.This indicator was supported by the indirect flow-related properties, these include angle of repose, Carr's index and Hausner ratio.All the formula showed the Hausner ratio below than 1.25.The result suggest that the C. mangga granule mixture showed good flowing characteristic.The Carr's index below than 16% showed free-flowing property [15].The Carr's index of all of the formula demonstrated value in the range of 10.26±3.38 to 17.54±1.60.According to Carr category on Carr's index and Hausner ratio [19], F3 and F6 revealed excellent flow properties; meanwhile, F1, F2, F4 and F5 demonstrated good flow property.Of all the formula, F2 showed the highest Carr's index, indicating that F2 had less free-flowing but still in good flowing category [19].All the formula showed the angle of repose in the range of 20.29±1.80 to 23.58±1.21,which were still below than 30 °indicating good flow ability [20].Capsule evaluation in terms of weight uniformity and disintegrating time are required to ensure that the capsules produced are consistent and homogeny.The weight uniformity is one parameter to calculate the weight of each capsule content whereby indicating the consistency of the dose distribution in each capsule.In the capsule evaluation result, it was showed that only F2 had fulfilled the Indonesian Pharmacopoeia requirement for weight uniformity.These phenomena could be influenced by several factors, such as the homogeneity of the granule mass and the accuracy of the filling process.Therefore, only the formula that met the Indonesian Pharmacopoeia capsule evaluation should be used for the animal study to evaluate the effectiveness of C. mangga extract as immunostimulatory in capsule dosage form.The C. mangga capsule of F2 formula was proceeded to animal study.
An in vivo investigation utilizing Wistar rats infected with S. aureus was conducted to assess the immunomodulatory properties of capsules containing dried ethanol extract of C. mangga rhizomes.To learn more about the immunomodulatory effects of C. mangga extract on DTH response and antibody generation, in vivo investigations on immune response were carried out.S. aureus was injected into every animal used in this study.Rats with S. aureus infections had their paw volume increased by the C. mangga capsule.The increase in paw edema is a sign that the rat immune system's DTH response has been stimulated.The result aligned with our previous studies, which showed that C. mangga extract stimulated phagocytosis activity [10].The examination into the development of antibodies also showed that C. mangga capsule boosted IgG production in rats.When C. mangga capsule was administered at a dose of 400 mg/kg bw, it showed the greatest activation of the DTH response and IgG production among all the samples.

CONCLUSION
The granule mass of C. mangga dried extract shows good flow ability property, as well as the capsule formulation of F2 met the requirement of Indonesian Pharmacopoeia.Furthermore, the capsule containing dried extract of C. mangga was able to stimulate the immune response, especially on the DTH response and antibody production.The results indicate that C. mangga capsule has the potential to be developed into a marketed immunomodulating product.However, further studies are required to evaluate the effect in humans by clinical study.