Synthesis And Comparative Evaluation Of Traditional Herbomineral Nanoceuticals For Their Haemopoietic Activity

The traditional System of medicine comprises a wide variety of formulations for various ailments. It is essential to Standardize Ayurvedic drugs so that drugs having good quality can be used for better effect. Standardization is not only limited to the (cid:977)inal preparation, but it also includes the materials used during the development and SOP adopted and the equipment used. The present study is an attempt to prepare anti anaemic herbomineral nanoceuti-cal by two different processes, and to standardize and evaluate the haemopoietic effect of the prepared formulations and compare with a marketed formulation. Preparation was done based on Ayurveda Pharmacopoeia. Formulations were characterized by conventional methods and by AAS, XRD, EDAX, SEM, TGA, and IR. The toxicity and anti-anaemic effectiveness are evaluated by using albino Wister rats and histopathology and cytotoxicity studies. Products have a crystalline nature (XRD). TGA shows the decomposition temp of ferric oxide. SEM EDAX revealed surface smoothness and nano to the micro size of particles. AAS shows that the elements are within the prescribed limits. The in-process Standardization shows the effectiveness of adjuvants. There is an increase of haemoglobin and body weight on day 75 for (cid:977)irst preparation than the Second and were comparable to that of standard. No toxicity in all rats and indicated no renal and gastrointestinal toxicity. The size, smoother surface, and the therapeutic effectiveness of the herbominerals prepared by the two methods can be attributed to the calcination steps and adjuvants. Thus, maybe dose reduction and rapid effect can be achieved.


INTRODUCTION
Even though modern medicine has achieved heaps and developed newer technology, most of the rural and remote areas around the world have got a keen interest in alternative Systems of medicines focusing on herbal medicine as the primary remedy for treating ailments. In India, the traditional medicinal systems comprising of Unani (The Arabian System), Ayurveda, and Siddha are famous for their effectiveness, and the present System of therapy has acknowledged the uniqueness of such a system of medicine. Many of these systems are practised from the Vedic period and are older than allopathic Systems of medicine (Kale and Rajurkar, 2019). There are several lacunae for mainstreaming of such medicine. The most important one is the lack of Standardization of the practising formulations, which is essential for quality control and the optimization of different parameters (Sen and Chakraborty, 2017;Bhatambreyogesh and Pallavi,  Dark red. With small amount of dull white color in between.         The process of Standardization of any drug comprises of 1. Screening of Raw materials for ensuring their quality 2. Standardization during the processing of Formulation Here evaluation of the procedure and the intermediates formed during its preparation is done by doing speci ic tests (Drugs and Cosmetics, 2009).

Formulation Standardization
Here, the inal product or the prepared medicine is evaluated as per guidelines (Prashant, 2019;Singh et al., 2016).
Ayurvedic formulations are prepared with many additives, which improve the therapeutic ef iciency and are expected to reduce the toxicity. The steps involved in the processing also affect quality. Bhasma is one of the formulations used in Siddha and Ayurveda under Rasoushadhi. Bhasma is prepared by a unique process known as Bhamikaran involving Sodhana, Bhavana, and Marana. The particular process makes smooth nano-sized particles having a very potent therapeutic effect with a reduced dose. Bhasmas are unique in their properties, and these metal-containing nanomaterials are obtained by repeated heating and are administered together with plant juices/fruit juices. These types of ayurvedic formulations are prescribed for the treatment of a variety of diseases. Usually, the adjuvants like honey, butter, lime juice, milk ghee used may help the metal early assimilable and may reduce the harmful effect, and thereby biocompatibility is increased (Rasheed, 2016;Rasheed and Shivshankar, 2015). Kaseesa bhama is a famous herbs mineral formulation used in Ayurveda for its anaemic anti action. It contains iron metal in oxide form. Since the method of preparation affects the quality of the inal product, the present study aims to standardize the process by selecting two different adjuvants for the Formulation of Kaseesa bhasma. It also includes the evaluation of the product by new analytical parameters and hemopoietic activity by using an animal model. The inal products compared with the marketed formulation (Mukkavalli et al., 2017;Rasheed, 2014).

MATERIALS AND METHODS
The critical ingredient of Kaseesa bhasma is ferrous sulphate powder, and it was procured from Nice Chemicals, Mumbai, India. Marketed Kaseesa bhasma and the other ingredients required were brought from the local market of Kannur district, Kerala, India. The puri ication (swedhana) process was done by using plants obtained from the herbal Garden of Pariyaram Ayurveda Medical College, Kannur, India, and duly authenticated.

Preparation
Kaseesa Bhasma prepared as per the Ayurvedic Pharmacopoeia. The steps involved are Shodhana/Puri ication, Bhavana/size reduction, and Puta/Calcination. Two different methods adopted for the Formulation of Bhasma by using different adjuvants (Elahi et al., 2017;Ayurvedic Pharmacopoeia, 2008).

Method 1(Formulation 1:KaB1)
Shodhana was done by taking Ferrous sulphate 250gm and using Bringaraja Swarasa of pH 7.5 ( 2L) and then heating for 3hrs. The compounds were made into a paste by trituration using a vessel known as khalvam and a smooth pate of Ferrous sulphate. Further, the mixture was made into a soft powder by Marana process to make and Chakrikas of dimension: diameter: 1.5 -2 cm. Thickness: 0.5 cm was prepared and transferred to a crucible named Sharava, and it is then covered and sealed by using sealing clay. The sharava is heated using Coconut husks for calcination of the contents. The calcination steps were repeated four times for 3hr, 4hr 2.15hrs, and for 2hrs respectively to get 20gm of product. The whole procedure repeated three times.

Method 2 (Formulation 1:KaB2)
Ferrous sulphate250gm then triturated and dried under sunlight for one day(Puri ication). Then using Nimbu Rasa 15ml, the compounds then made into a paste by keeping in khalvam. The obtained mixture powdered (Marana) Chakrikas of dimension: diameter: 1.5 -2 cm. Thickness: 0.5 cm was prepared and transferred to a crucible named Sharava and then covered and sealed as method I. Finally, calcination has done by heating (Cow dung 75 Kg a fuel). The calcination steps were repeated for four times for 3hrs to get 20gm of product. The whole procedure repeated three times [Rasatharangini] (Sharma, 2014).

Preparation Characterization
The formulations KaB1and KaB2 was subjected to evaluation by the traditional method (Rasheed and Shivashankar, 2017;Wadekar et al., 2006).
The prepared formulations were standardized initially by conventional means, which involve loating property determination, testing ineness, and metallic lustre for the preliminary analytical con irmation of Bhasma preparation. Physical evaluations like colour, odour, taste and pH and the determination of Ash values and LOD were also evaluated for the formulations and compared with the standard mar-keted Formulation (Wadekar et al., 2005;Sharma et al., 2016).
Analytical standardization involves IR, XRD analysis, after each stage of Formulation. The XRD pattern is used for determining the crystalline size using the Scherrer equation (Bhange, 2017;Madhavan et al., 2016).
The inal Formulation further subjected to instrumental analysis like AAS, TGA, SEM, TEM (Sharma et al., 2009). Quantitative determination of iron present in the formulated bhasma was done by using Atomic Absorption Spectrometer (Perkin Elmer, USA) (Chavare et al., 2017;Pal, 2015).

2.3
The therapeutic ef iciency of formulations was checked for hematopoietic effect using albino Wistar Rats by inducing anaemia by phlebectomy

Animals
Male Wistar albino rats weighing 90-100g, aged about 45 ± 2 days, were kept separately in polypropylene cages at a temperature of 25 ± 10 ºC with average humidity ranging from 45-55 % and day and night cycle of 12:12 hrs. Standard rat pellets and water ad libitum fed to each rat of treatment groups. A period of 48 h acclimatization to the laboratory conditions made before the commencement experiments to experimental rats. Animal experiments were done with the approval and the guidelines as per IAECP (Sumithra et al., 2015;Luthy et al., 2009).

Antianemic ef icacy
Male Wistar albino rats grouped as anemic and nonanemic were the treatment groups for the study. The anemic group of rats comprised of further four groups for the treatment schedule. Each group consists of six animals. Agar gel diet, prepared according to the standard method, were fed to the animals of the anemic group. The diet was changed every 7 days and restricted to once a day. The diet was deprived of any trace of iron in it, and water was supplied ad libitum only after triple distillation to avoid any traces of iron contamination to the treated animal. Non-anemic group rats were given with a measured quantity of ferrous sulfate apart from an agar gel diet and suf icient water. Anemia was induced by phlebotomy (Sivakumar et al., 1985). The agar gel diet was given continually for 15 days, and then phlebotomy was performed on 1 st , 4th, 8th, and 10th day. 0.6 ml of blood were bleed each day. The treatment schedule for the four anemic groups was as follows

Group III
Marketed Formulation of Kaseesa bhasma, KaB3 (0.02 mg/kg) in an aqueous medium

Group IV
Vehicle control used was a suspension of 2% gum acacia All the treatments to each of the animals were orally given using feeding cannula and were given daily on the ifteenth day of starting the agar gel diet schedule and prolonged the treatment up to 30 days. Untreated control groups were the Nonanemic groups who had not fed with any drug. The standard method for human dosage used for calculating the dose of different treatments given to the anemic group. (Bennett et al., 2012). On every 15 th day, the body weight (in g) of animals in each group calculated.

Estimation of Hemoglobin
The cyanomethemoglobin method used to determine hemoglobin level each animal from the blood collected on 0, 4, 8, 10, 12, 30, 45, 60, and up to the 75 th day of the commencement treatment (Rasheed, 2014).
Determination of Serum ferritin: ELISA reader and conventional method were used for the estimation of serum ferritin level in every animal of each group by using the serum collected on 15 th and 75 th day

Determination of Serum iron and total ironbinding capacity (TIBC)
On the 15 th and 75 th day, the blood serum collected from each animal of each group evaluated using spectrophotometry at 562 nm for estimating the concentration of Serum iron and TIBC (Kaur and Kawatra, 1994).

Evaluation of Toxicity
Toxicological studies were performed using 0.02 mg/kg as the starting dose Kaseesa bhasma. To overnight fasted rats, formulations were administered orally. After the administration also the food was withdrawn for 3-4 h and the animals were observed for any toxicity signs. It includes a reduction in body weight and any marked difference in eyes, mucous membranes, skin and fur, and in respiratory, ANS, and CNS and circulatory Systems. Pharmacological parameters, like motor activity, salivation, sleep, and coma, were also observed. OECD 423 guideline has followed for the entire studies Toxicity of formulations was further screened by Histopathological studies of tissues from the stomach, liver, and kidney. The study was done by isolating the organs immediately after scari ication, and the stomach, kidney, and liver tissues were separated and ixed in 10 % formalin. Training done by using routine hematoxylin and eosin and sections were of 4-5 micron thickness. The histopathological sections examined with the cooperation of consultant histopathologist examination and images of sections were taken with optical microscope (Pattanaik et al., 2003;Pandit et al., 1999).

Statistical analysis
The signi icance of the study assessed by one way ANOVA analysis; the signi icance was evaluated for observations with p values less than 0.05.

Observations And Reports
Physical characterization

Formulation 1(KaB1)
The adjuvant Bringarajawarasya has reduced its pH from 7.25 to 4.49 after swedana on heating for 3hrs to an average temperature of 454 0 C. The temperature and appearance of Kaseesa after each calcination was different.
[ Table 1] The color of the product changed from pale green to red.

Formulation 1(KaB2)
The adjuvant Nimbu Rasa has reduced the color from light green to yellow [ Table 1].

Preliminary Evaluation
The stepwise processes adopted for the Formulation of Kaseesa bhasma KaB1 and KaB2 were as per Figures 1 and 2. The traditional method of characterization for KaB1and KaB2 showed them with brick red color, not having any taste, with a characteristic smell and having a pH of 7.4 [ Table 2]. The Lightness formulations proved to sprinkle the bhasma over the water, and it has shown loating over the surface. Bhasma particle entry into the groves of ingers when rubbed indicates the ineness. The bhasma showed no luster or brightness when exposed to sunlight, indicating that the metal has loosed its metallic shine. The marketed Formulation and KaB3 as well showed characteristics similarly.
The physio-chemical experiments for KaB1, KaB2, and KaB3 were different Ash values, and loss on drying performed. The results obtained were as per Table 3.

Analytical standardization
Analytical Standardization involves FTIR, XRD analysis of Formulation of KaB1, KaB2, and KaB3 (Fig-ures 3 and 4). The results show that the amorphous nature of particle changes to crystalline one after the preparation has completed. The FTIR spectroscopy plot does not show any peak for the presence of any organic molecule and that of any organic moiety bond.
Comparative analysis of XRD results demonstrates the characteristic re lection peaks of all the three samples, and all were at identical values. XRD pattern shows the sharp, high intensity of lines, which indicates that the chemical moiety is existing in crystalline form. The crystalline size calculated from each of the XRD patterns plotted and Scherrer formula used. The value was in the range of 53 -57 nm. The peaks in the spectra correspond to that of Fe 2 O 3 form and in hydrated metal oxide (FeO or Fe 2 O 3 ) of inorganic iron. From the AAS study was the concentration of elements seen in all three formulations was detected. From the results, it revealed that the iron was the element present in signi icant concentration with 65.54 %, 68.92%, and 79.91 % for KaB1 and KaB2 and KaB3, respectively, and the other entire element are within the speci ied limits (Table 4).
SEM images of KaB1, KaB2, and KaB3 are given in Figure 5 indicates size variations and particle agglomeration within the formulations. Repeated calcination cycles involved in the preparation is responsible for the agglomeration. Depending upon the temperature achieved during heating and the adjuvants used, the particle size and agglomeration may occur. In KaB2 with lemon juice adjuvant, ferric oxide shows well-de ined structures while the KaB1showed spongy, microcrystalline aggregates without marked boundaries. The large particle size of KaB2 and KaB3 than KaB1 can be attributed to the preparation methodology that includes several calcination steps. The method adopted for preparation, calcination temperature and duration of preparation may in luence the morphology and were reported in previous studies.
TGA curves were obtained for all the preparations of Kaseesa Bhasma by heating in air and were obtained as given in Figure 6. The complete decomposition of KaB1 and KaB2 and KaB3 formulations were occurred at 1210 ºC 1232 ºC and 1279 ºC respectively. Heating of ferrous sulphate in air converted the compound to ferric oxide. As the TGA curves showed the complete decomposition at1210 ºC, 1232 ºC, 1279 ºC respectively therefore con irms the presence of ferric oxide.

Pharmacological standardization
In this study, Kaseesa Bhasma KaB1 showed an appreciable increase of hemoglobin (13.01 %) on the 75th day, and KaB2 showed an increase close to that of marketed Formulation treated group (13.70 %) indicating prominent Anti-anemic activity (Table 5). Rats of Bhasma treated groups showed a signi icant rise in average bodyweight that was comparable to the standard non-anemic group ( Table 6). As loss of body weight is the signi icant clinical indications of iron de iciency anemia, it may conclude that treating with Kaseesa bhasma may help in overcoming the weight loss.
The serum ferritin level is determined to assess the total available iron, which is essential to determine the anemic state. As the stored iron is high in the formulated and standard bhasmas, the increase in serum ferritin level indicates that Kaseea bhasma can be used effectively in iron de iciency anemia as well as for other iron-de icient diseases. The serum ferritin level is comparatively more in KaB1 Compared to the other two Bhasmas. The clinical study on iron de iciency anemia showed that the binding of serum proteins with the free iron and the TIBC (Total iron binding Capacity) increases as the disease progress. This work shows that the treatment with Kaseesa Bhasmas understudy has produced a signi icant decrease of TIBC level, which indicates the role of bhasma in the dissociation of bound iron from serum protein and thereby producing an increase in iron level in serum (Table 7). When compared to the result, the KaB1 slightly better than KaB2 and marketed Bhasma. Another signi icant result found was the in lation level of serum iron in KaB1 KaB2 and Marketed Bhasma, and that attributed to their effectiveness in iron de iciency anemia.

Toxicological studies
KaB1 KaB2 and Marketed Bhasma had not showed any toxicity symptoms in any of the treated rats and constant body weights was noted before and after the administration of formulations and that indicated the absence of renal and gastro intestinal toxicity for the Kaseea Bhasma (Table 8). The histopathological sections shown in Figure 7 does not revealed any signs of toxicity in liver and kidney cells.

CONCLUSIONS
The main component of Kaseesa bhasma found as iron. The various stages of formulation techniques involved, like shodhana (puri ication) Marana [consisting of Bhavana (wet trituration) and the calcination process (puta), reduces the particle size remarkably, and thereby improved absorption, metabolism, and distribution of the drug into the various systems of the body. By the analytical tech-niques studies, the size of the formulations revealed as micro to the nano range. From the indings of the present study, we can conclude that Kaseesa Bhasma effectively used for the treatment of iron de iciency anemia, and the method of preparation has a signi icant role in the absorbability of the Formulation. Out of the three formulations under the studies, KaB1 has shown comparatively more haemopoietic activity. The reason may be attributed to the adjuvant used (Bringaraja swarasa) and the process of preparation. This work is an attempt to carry out in-process Standardization for Herbometallic nanoceuticals.

Ethical Issues
The study was approved by the CPSEA