DESC: FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENTS RULES, 2003
(See section 10; rule 13)

HERBAL DRUG COMPOSITION, ITS PREPARATION PROCESS AND USE THEREOF

NAME AND ADDRESS OF THE APPLICANT:

Savitribai Phule Pune University
An Indian Entity, having address as:
Ganeshkhind, Pune 411007, Maharashtra, India

The following specification particularly describes the invention and the manner in which it is to be performed.

CROSS-REFERENCE TO RELATED APPLICATIONS
The present application claims priority from Indian Patent application 202021023652 filed on 05-Jun-2020
TECHNICAL FIELD
The present disclosure generally relates to an anti-sickling agent, and more particularly, to a herbal formulation for health management in Sickle Cell Disease. The disclosure further relates to a method for preparing a herbal formulation for health management in Sickle Cell Disease.
DESCRIPTION OF THE RELATED ART
Sickle cell disease (SCD) is a group of hereditary blood disorders. SCD is characterized by red blood cells that assume an abnormal, rigid, sickle shape. Healthy red blood cells are round, and they move through small blood vessels to carry oxygen to all parts of the body. For a sickle cell diseased patient, their red blood cells die early, causing a constant shortage. Also, when these sickle cells travel through small blood vessels, they get stuck and clog the blood flow, this causes excruciating pain and other complications. In SCD, Red Blood Corpuscles reveal a reduced oxygen carrying capacity and decrease life span to 10-20 days. The generation of abnormal sickled erythrocytes leads to inconstant degree of hemolytic anemia, acute and chronic tissue damage due to vaso-occlusion. The term sickle cell anemia is reserved for a homozygous state (Hb S) for sickle cell gene.
In India, the prevalence of SCD is mainly distributed in states of Madhya Pradesh, Chhattisgarh, Maharashtra, Orissa and Andhra Pradesh. It is the second most common hemoglobinopathy, after to Thalassemia. As per an ICMR survey, about 20% of children with SCD expired by age of two and 30% of children with SCD among the tribal community die before they reach adulthood.

Treatment of sickle cell disease has proved difficult and inefficient due to its genetic origin. Earlier therapies included use of liver-based extracts and diets, oxygen vasodilator, carbonic anhydrase inhibitors, and splenectomy. Other conventional therapeutic management involves the use of drugs such as piracetam, tucaresol, hydroxyurea as well as bone marrow transplantation and gene therapy. The major side effects of Hydroxyurea include decreased production of platelets, red blood cells and white blood cells. Bone marrow transplantation may give dramatic results, but its routine use is not possible because of the complications associated with the procedure, highly sophisticated infrastructure and appropriate donors besides the high cost. A technique like medullar transplantation and blood transfusion are costly and out of reach of the masses which may also expose the patient to mutagenicity, iron overload and other fatal risks.
Sickle cell disease is an international health problem and truly a global challenge. Hence there is a need to find out an ideal therapy which is potent safe and cost effective.
BRIEF DESCRIPTION OF THE DRAWINGS
The embodiments herein will be better understood from the following detailed description with reference to the drawings, in which:
FIG. 1 is a flow diagram illustrating a method for preparing a herbal drug composition as an anti-sickling agent for treatment of Sickle Cell disease according to an embodiment herein;
FIG. 2 is a flow diagram illustrating a method for analyzing the quality of the herbal drug composition according to an embodiment herein;
FIG. 3 illustrates a graphical representation for inhibition of polymerization of the Sickle hemoglobin protein (Hb S) according to an embodiment herein;
FIG. 4 illustrates a comparative graphical representation for increase in Fe2+/Fe3+ ratio according to an embodiment herein;
FIG. 5 illustrates a graphical representation for osmotic fragility of the herbal drug composition according to an embodiment herein;
FIG. 6 illustrates a comparative representation for reversal of sickling test according to an embodiment herein;
FIG. 7 illustrates a graphical representation for antioxidant acitivity of the herbal drug composition according to an embodiment herein;
FIG. 8 illustrates a graphical representation for comparative study of polymerization inhibition of the Sickle hemoglobin protein (HbS) using 13 herbal drug compositions.
FIG. 9 illustrates a table of comparative study to identify an appropiate composition of the herbal drug composition in the effective management of the sickle cell disease according to an embodiment herein.
Figure 10 shows the LCMS - Total Ion Chromatogram of the herbal drug composition according to an embodiment herein.
Figure 11 shows the HPLC chromatogram of herbal drug composition according to an embodiment herein.
Figure 12 shows the HPTLC fingerprint of herbal drug composition according to an embodiment herein.
Figure 13 shows the photomicrographs of kidney and liver sections Hematoxylin and Eosin (H & E) staining after acute oral toxicity.
Figure 14 shows the photomicrographs of kidney and liver sections ( H & E staining) after subacute toxicity study.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
The embodiments herein and the various features and advantageous details thereof are explained more fully with reference to the non-limiting embodiments that are illustrated in the accompanying drawings and detailed in the following description. Descriptions of well-known components and processing techniques are omitted so as to not unnecessarily obscure the embodiments herein. The examples used herein are intended merely to facilitate an understanding of ways in which the embodiments herein may be practiced and to further enable those of skill in the art to practice the embodiments herein. Accordingly, the examples should not be construed as limiting the scope of the embodiments herein.
As mentioned, there is a need to find out an ideal therapy for Sickle Cell disease which is potent, safe and cost effective, refraining from the side effects of other treatments and method. In the present invention herbal drug composition is a novel herbal composition prepared from unique medicinal plant extracts which acts as an antisickling agent, which are safe and has no side effects. The herbal drug composition increases oxygen affinity of hemoglobin protein (Hb S) and membrane stability of Sickle Cell RBCs, prevents the oxidative stress produced in the Sickle Cell RBCs due to its antioxidant property thereby increasing life span of the Sickle Cell RBCs. Overall, the herbal drug compostion leads to decrease in pathophysiological conditions and provides benefits for lethal manifestation in Sickle Cell Disease. Health benefits of the herbal drug composition includes but not limited to antioxidants, anti-inflammatory, hepatoprotective, anti-bacterial, antifungal and anticancer activities. The herbal drug compostion also contains various pharmacologically active phytochemical compounds which may individually or synergistically be effective in the overall health management of the Sickle cell disease in which multiple organs or sites get affected.
Referring now to the drawings, and more particularly to FIGS. 1 to14, where similar reference characters denote corresponding features consistently throughout the figures, preferred embodiments are shown.
FIG. 1 is a flow diagram illustrating a method for preparing the herbal drug composition as anti-sickling agent for treatment of Sickle Cell disease according to an embodiment herein; At step 101 and 102, medicinaly important plant materials are selected on the basis of its medicinal value other than antisickling property, easy availability, least toxicity and cost effectiveness, and is purchased from GMP certified company “Green Pharmacy, Pune”. An aqueous extract of the plant material is prepared. The aqueous extract of the Thinopyrum intermedium (Wheatgrass), the Punica granatum (Pomegranates peel), the Rubia cordifolia (Indian madder stem), and the Aegle marmelos (Indian bael leaf) is prepared using decoction technique. The prepared aqueous extract of plant materials, may include but not limited to types of solvent used for extraction of Thinopyrum intermedium (Wheatgrass), Punica granatum (Pomegranates peel), Rubia cordifolia (Indian madder stem), and Aegle marmelos (Indian bael leaf) which have medicinal properties. Said types of solvent is solvent selected from the group consisting of methanol, ethanol, hydro-alcohol, ethyl acetate, acidified aqueous solution, saline water or buffer solutions. The aqueous extract of plant materials is selected after screening of various medicinal plants for their anti-sickling, easy availability and cost effectiveness. The aqueous extract of each plant material enclosed herewith has individualy good in-vitro anti-sickling properties recorded and polymerization inhibition of the Sickle hemoglobin protein (HbS) as compared to hydroxyurea (HU) indicating its potential as antisickling agent, wherein the hydroxyurea is a standard drug in treatment of the sickle cell disease.
At Step 103 the aqueous extract of the plant materials is filtered to obtain a filtrate of the aqueous plant extract. At step 104 the filtrate of the aqueous plant extract is dried using at least one but not limited to spray drying, high vacuum evaporation or lyophilisation to prepare a solid plant extract. At step 105 the solid plant extracts from the plant materials are collected in equal proportion to prepare the herbal drug compostion.
In another embodiment, the extract is prepared from any other solvent selected from the group consisting of methanol, ethanol, hydro-alcohol, ethyl acetate, acidified aqueous solution, saline water or buffer solutions followed by the steps of filtration and drying to obtain a solid plant extract.
In one embodiment the composition of 25% w/w of the plant extracts wherein the extract is an aqueous extract or any other solvent extract selected from the group consisting of methanol, ethanol, hydro-alcohol, ethyl acetate, acidified aqueous solution, saline water or buffer solutions are mixed to prepare the herbal drug composition.
In one embodiment, the herbal drug composition has good anti-sickling activity in terms of polymerization inhibition of sickle haemoglobin protein in hypoxic condition. The herbal drug composition has also ability to increase oxygenated hemoglobin indicated by the study of Fe2+/Fe3+ ratio and most essentially an improved membrane stability of Sickle Cell RBCs. Furthermore, the reversal of sickling bioassay strengthens the anti-sickling potential of the herbal drug composition.
In another embodiment, the herbal drug composition has individual or synergistic pharmacological active phytochemical compounds for overall management of sickle cell disease in which multiple organs or sites get affected. In yet another embodiment the herbal drug composition can be manufactured in the form of tablets, capsules, syrup or any suitable form of medicine for oral administration using available preservatives or additives.
FIG. 2 is a flow diagram illustrating a method for analyzing the quality of the herbal drug composition according to an embodiment herein; At step 201 the herbal drug compostion is investigated for their anti-sickling properties. At step 202 phytochemical and metabolomics profile of the herbal drug composition is analysed using HPTLC, HPLC, and LC-MS. At Step 203, in-vivo Preclinical Biosafety evolution of the herbal drug composition is performed on rat model.
In one embodiment, the phytochemical analysis of the herbal drug composition is carried out using advance techniques such as HPTLC, HPLC, and LC-MS equipped with plant metabolite mass spectral library. The identified compounds in the herbal drug composition have individual and synergistic pharmacological effect for overall management and treatment of pathophysiological conditions prevalent in the Sickle Cell Disease patients. In another embodiment, in-vivo acute and sub-acute toxicity study on rat model is tested for biosafety of the herbal drug composition.
FIG. 3 illustrates a graphical representation for inhibition of polymerization of the Sickle hemoglobin protein (HbS) according to an embodiment herein. The graphical representation 300 includes the percentage of inhibition of polymerization individually of each plant extract and the herbal drug composition. The herbal drug composition showed higher polymerization inhibition of the Sickle hemoglobin protein (Hb S) as compared to each of the plant extracts. In one embodiment the polymerization inhibition of the Sickle hemoglobin protein (Hb S) of the herbal drug compostion can be not limited to 79.41%.
FIG. 4 illustrates a comparative graphical representation for increase in Fe2+/Fe3+ ratio according to an embodiment herein. ‘A’ of the graphical representation 400 shows a percentage of Oxygenated Hemoglobin (HbO2), ‘B’of the graphical representation 400 shows a percentage of Met Hemoglobin and ‘C’ of the graphical representation 400 shows a percentage increase in the Fe2+/Fe3+ ratio. The herbal drug compostion showed an increased percentage of the Fe2+/Fe3+ ratio, indicating increased affinity to oxygen by the Sickle Hemoglobin protein (HbS). The graphical representation 400 proves an enhanced oxygen carrying capacity of the Sickle Hemoglobin protein and thereby useful for hypoxia in Sickle Cell patients due to the use of the herbal drug compostion.
In one embodiment the increase in the percentage of Fe2+/Fe3+ ratio can be but not limitd to 86.98%. which is effective for hypoxia in the Sickle Cell patients.
FIG. 5 illustrates a graphical representation for osmotic fragility of the herbal drug composition according to an embodiment herein. The graphical representation 500 represents a concentration of NaCl on the x axis and percentage of hemolysis on y axis. ‘A’ includes the percentage of hemolysis by Phosphate Buffer saline (PBS). ‘B’ includes the percentage of hemolysis by the herbal drug composition and ‘C’ includes the percentage of hemolysis by standard drug the HU. The herbal drug compostion shows highest reduction of the percentage of hemolysis thereby increasing membrane stability of Sickle cell RBCs as compared to the HU and the PBS as a control. The life span of sickle RBCs has increased due to the increase membrane stability after the application of the herbal drug composition at various physiological osmotic stress.
In One embodiment the herbal drug composition protects the Sickle cell RBCs from different osmotic stress and increases life span and reduces the overall crisis attempts in the Sicle Cell Disease.
FIG. 6 illustrates a comparative representation for reversal of sickling test according to an embodiment herein. ‘A’ represents the result of reversal of sickling test with Phosphate Buffer saline (PBS), ‘B’ represents the result of reversal of sickling test through the standard drug HU and ‘C’ represents the reversal of sickling test by the herbal drug composition. The percentage of reversal of sickling is highest with the herbal drug compostion. The reversal of sickling test confirms the herbal drug compositon as an antisickling agent. The herbal drug composition exhibits reversal of sickle cell RBCs to normal shape. In one embodiment the herbal drug compostion shows a good reversal of sickling which is not less than 75% as compared to control the PBS and as effective as standard drug the HU.
FIG. 7 illustrates a graphical representation for antioxidant activity of the herbal drug composition according to an embodiment herein. The antioxidant activity of the herbal drug compostion is 98.25 mg/g ascorbic acid equivalent. The herbal drug composition shows a higher antioxidant potential for protecting oxidative damage in the sickle cell disease. The protection from the oxidative damage is assessed using DPPH radical scavenging activity as compared to ascorbic acid as standard.
FIG. 8 illustrates a graphical representation for comparative study of polymerization inhibition of the sickle hemoglobin protein (Hb S) using 13 herbal drug compositions represented by “a to m” according to an embodiment herein. The 13 different composition of the herbal drug compostion is analysed using standard drug the Hydroxyurea (HU). The equal composition of 25% w/w of the solid plant extracts showed highest polymerization inihibition of the sickle hemoglobin protein (Hb S). The highest polymerization inhibition of the sickle hemoglobin protein (Hb S) signifies as a potential anti-sickling agent.
FIG. 9 illustrates a table of comparative study to identify an appropiate composition of the herbal drug composition in the effective management of the sickle cell disease according to an embodiment herein. The table illustrates a combination of 13 different compositions represented by “a to m” for the herbal drug composition to identify the most appropriate composition to make the herbal drug wherein w1 represents Thinopyrum intermedium (Wheatgrass); w2 represents Punica granatum (Pomegranates peel), w3 represents Rubia cordifolia (Indian madder stem) and w4 represents Aegle marmelos (Indian bael leaf).
In one embodiment, the composition of 25% (w/w) is identified to be a suitable composition for the herbal drug. In another embodiment the 13 different composition of the herbal drug compostion is analysed using standard drug the Hydroxyurea (HU). The equal composition of 25% w/w of the solid plant extracts showed the highest polymerization inihibition of the sickle hemoglobin protein (Hb S). The highest polymerization inhibition of the sickle hemoglobin protein (Hb S) signifies as a potential anti-sickling agent.
In one embodiment the phytochemical analysis of the herbal drug composition is carried out using advance techniques such as HPTLC, HPLC and LC-MS equipped with plant metabolite mass spectral library.
LC-MS STUDY:
Sr. No. Name Formula m/z RT Score (DB)
1 pentan-2-one C5 H10 O 104.107 0.67 99.94
2 2S-amino-pentanoic acid C5 H11 N O2 140.068 0.68 87.57
3 L-2-Amino-4-(hydroxymethylphosphinyl)butanoate C5 H12 N O4 P 182.0574 0.68 85.11
4 carisoprodol C12 H24 N2 O4 283.1627 0.69 99.22
5 a-D-Glucose C6 H12 O6 203.0522 0.73 86.39
6 N-Acetyl-leucyl-leucine C14 H26 N2 O4 287.1964 0.79 85.81
7 1-Aminocyclohexanecarboxylic acid C7 H13 N O2 144.1018 0.82 99.94
8 2-ethyl-hydracrylic acid C5 H10 O3 118.0862 0.84 99.79
9 3-Oxovalproic acid C8 H14 O3 158.117 0.85 85.49
10 Hexylamine C6 H15 N 102.1276 0.85 87.31
11 Zizybeoside I C19 H28 O11 432.1862 0.88 99.51
12 N-(3-oxo-octanoyl)-homoserine lactone C12 H19 N O4 241.1544 0.91 85.95
13 3D-(3,5/4)-Trihydroxycyclohexane-1,2-dione C6 H8 O5 143.0338 0.94 87.46
14 Valproic acid C8 H16 O2 144.1379 0.95 99.2
15 Hygrine C8 H15 N O 124.1119 0.99 87.66
16 Lamprolobine C15 H24 N2 O2 247.1804 1.19 87
17 4-Hydroxystyrene C8 H8 O 120.0807 1.2 87.72
18 Normetanephrine C9 H13 N O3 166.0859 1.2 91.6
19 Nupharidine C15 H23 N O2 249.1959 1.2 94.1
20 trans-Cinnamoyl beta-D-glucoside C15 H18 O7 310.1284 1.26 98.78
21 1-Phenylethanol C8 H10 O 105.0697 1.46 87.15
22 (5R)-6-Hydroxy-5-isopropenyl-2-methylhexanoate C10 H18 O3 186.1488 1.74 99.8
23 Pseudoconhydrine C8 H17 N O 126.1276 1.74 99.73
24 DL-Indole-3-lactic acid (3-Indolelactic acid) C11 H11 N O3 188.0703 1.88 97.62
25 6-Hydroxydopamine C8 H11 N O3 152.0704 1.89 98.37
26 Flutriafol C16 H13 F2 N3 O 301.1257 1.94 86.63
27 Lophophorine C13 H17 N O3 235.1438 2.03 94.22
28 p-Coumaroylagmatine C14 H20 N4 O2 277.1656 2.39 96.74
29 N,N-Diethylglycine C6 H13 N O2 114.0911 2.47 85.98
30 Salicyl alcohol C7 H8 O2 107.0493 3.49 87.47
31 3,4-Dihydroxystyrene C8 H8 O2 119.0489 3.76 86.7
32 o-Coumaric acid C9 H8 O3 147.0439 3.76 86.03
33 Vanilpyruvic acid C10 H10 O5 193.0496 3.77 85.87
34 Aspirin C9 H8 O4 163.0386 3.95 86.06
35 Loganin C17 H26 O10 395.1308 4.88 99.09
36 Bakankoside C16 H23 N O8 362.1202 5.1 97.81
37 17beta-(Acetylthio)estra-1,3,5(10)-trien-3-ol acetate C22 H28 O3 S 373.1827 5.28 87.83
38 3-hydroxy-decanoic acid C10 H20 O3 211.1302 5.36 86.82
39 1-Octen-3-ol-3-o-beta-D-xylopyranosyl(1->6)-beta-D-glucopyranoside C19 H34 O10 427.1931 5.62 87.13
40 Queuosine C17 H23 N5 O7 409.1825 5.66 90.09
41 Lamioside C18 H28 O11 425.1415 5.67 99.26
42 Glucovanillin C14 H18 O8 337.0887 5.98 94.3
43 Tetrahydrogeranylgeranyl diphosphate C20 H40 O7 P2 493.1884 6.06 86.41
44 1-O-Sinapoyl-ß-D-glucose C17 H22 O10 391.0994 6.43 98.56
45 2,4,8-Trihydroxy-1-tetralone C10 H10 O4 177.0541 6.44 98.92
46 Meloside A C27 H30 O15 595.1646 6.56 97.33
47 Schizonepetoside E C16 H28 O8 371.167 6.6 97.79
48 Acanthoside D C34 H46 O18 765.2569 6.72 91.42
49 Netilmicin C21 H41 N5 O7 476.3058 6.75 90.86
50 Butrin C27 H32 O15 579.17 6.85 98.12
51 Kaempferol 3-O-ß-D-glucosyl-(1->2)-ß-D-glucoside C27 H30 O16 633.1419 6.88 99.12
52 4-t-Butylbenzoic acid C11 H14 O2 179.1063 6.98 86.42
53 Anthemis glycoside B C34 H41 N O17 735.2616 7 88.75
54 Fexaramine C32 H36 N2 O3 517.2247 7 86.5
55 2,2',3-Trihydroxy-3'-methoxy-5,5'-dicarboxybiphenyl C15 H12 O8 303.0494 7.01 99.14
56 Gossypetin 8-rhamnoside C21 H20 O12 487.0837 7.01 97.89
57 Quercetin 3-O-(6-O-malonyl-ß-D-glucoside) C24 H22 O15 551.1017 7.27 96.02
58 (±)-Taxifolin C15 H12 O7 287.0546 7.43 98.63
59 Plantagoside C21 H22 O12 471.0889 7.43 97.08
60 Dihydrofolic acid C19 H21 N7 O6 426.1517 7.48 96.24
61 Asperuloside tetraacetate C26 H30 O15 587.1361 7.49 94.91
62 (+)-Neomenthol C10 H20 O 174.1848 7.52 86.42
63 (2S,3S)-2-hydroxytridecane-1,2,3-tricarboxylic acid C16 H28 O7 355.172 7.52 98
64 Lauric acid C12 H24 O2 218.2111 7.53 99.47
65 Isobrucein A C26 H34 O11 545.1985 7.58 95.36
66 10-Deacetyl-2-debenzoylbaccatin III C22 H32 O9 445.1822 7.63 92.72
67 Narceine C23 H27 N O8 468.1619 7.65 97.24
68 Leucovorin C20 H23 N7 O7 456.162 7.69 88.77
69 Patrinoside C21 H34 O11 485.1986 7.78 91.12
70 beta-hydroxylauric acid C12 H24 O3 234.206 7.84 99.05
71 Mesuaxanthone A C14 H10 O5 241.0492 7.87 86.26
72 Eupatoroxin C20 H24 O8 397.125 7.9 94.12
73 Astemizole C28 H31 F N4 O 441.2449 8.1 93.98
74 Dianthalexin C14 H9 N O3 239.0811 8.16 98.47
75 MG(0:0/14:1(9Z)/0:0) C17 H32 O4 318.2635 8.18 98.91
76 Cnicin C20 H26 O7 383.1463 8.25 99.3
77 Zaragozic acid C C40 H50 O14 777.3085 8.27 93.2
78 Vomicine C22 H24 N2 O4 403.1621 8.29 89.45
79 Pretazettine C18 H21 N O5 314.1383 8.36 99.35
80 Genipin 1-beta-gentiobioside C23 H34 O15 571.1414 8.45 93.43
81 Carapanaubine C23 H28 N2 O6 433.1732 8.46 98.33
82 Glycyrrhizinic acid C42 H62 O16 843.3559 8.46 87.8
83 Cleomiscosin A C20 H18 O8 409.0886 8.84 96.08
84 Butopyronoxyl C12 H18 O4 249.1094 8.88 85.81
85 Soularubinone C25 H34 O10 517.2035 9.14 93.25
86 Debromohymenialdisine C11 H11 N5 O2 263.1252 9.22 86.4
87 Myristic acid C14 H28 O2 246.2426 9.32 99.08
88 Laurylaldehyde C12 H24 O 202.2162 9.35 94.9
89 2-Hydroxymyristic Acid C14 H28 O3 262.2372 9.53 97.6
90 UDP-3-O-(3-hydroxytetradecanoyl)-N-acetylglucosamine C31 H53 N3 O19 P2 833.2975 9.63 87.9
91 (S)-Coclaurine C17 H19 N O3 268.1328 9.8 85.36
92 4-Heptyloxyphenol C13 H20 O2 226.1799 9.88 93.69
93 3-Hydroxyestra-1,3,5(10),7-tetraene-16,17-dione 16-oxime C18 H19 N O3 280.1328 10.02 98.22
94 Oxycodone C18 H21 N O4 320.1255 10.02 85.5
95 3-tert-Butyl-5-methylcatechol C11 H16 O2 181.1219 10.12 85.22
96 Queuine C12 H15 N5 O3 277.1405 10.13 85.85
97 Arctiopicrin C19 H26 O6 355.1509 10.15 94.62
98 (10S)-Juvenile hormone III acid diol C15 H26 O4 293.1725 10.31 86.17
99 Neomenthyl acetate C12 H22 O2 198.1849 10.58 86.51
100 Furegrelate C15 H11 N O3 253.0971 10.67 86.61
101 Nonanoic acid C9 H18 O2 158.1538 10.68 99.68
102 Valeric acid C5 H10 O2 102.0909 10.68 86.57
103 Ngaione (-) C15 H22 O3 233.1533 10.72 85.7
104 C16 Sphinganine C16 H35 N O2 274.2739 10.86 99.58
105 alpha-Ionone C13 H20 O 175.1477 10.87 85.33
106 (+/-)-6-Hydroxy-3-oxo-alpha-ionol C13 H20 O3 225.1481 11 97.34
107 16-hydroxy hexadecanoic acid C16 H32 O3 290.2696 11.04 93.18
108 Palmitic Acid methyl ester C17 H34 O2 288.2897 11.05 86.52
109 12-trans-Hydroxy juvenile hormone III C16 H26 O4 283.1899 11.13 93.52
110 2-methoxy-hexadecanoic acid C17 H34 O3 286.2744 11.21 85.77
111 pentadecanal C15 H30 O 244.2643 11.34 95.03
112 Dillapiole C12 H14 O4 245.0787 11.43 99.34
113 tetradecan1-ol C14 H30 O 214.2534 11.49 85.94
114 Kikkanol D C15 H26 O3 237.1857 11.95 97.42
115 4,5ß-Dihydrocortisone C21 H30 O5 363.215 12.02 86.87
116 Sphinganine C18 H39 N O2 302.3055 12.27 85.67
117 cimigenol C30 H48 O5 511.3401 12.41 90.08
118 Decylubiquinol C19 H32 O4 325.2376 13.48 92.28
119 Glutinosone C14 H20 O2 243.1358 14.03 85.8
120 Euphornin C33 H44 O9 567.2932 14.06 91.15
121 Triphenyl phosphate C18 H15 O4 P 327.0777 14.26 90.86
122 10,11-epoxy-3,7,11-trimethyl-2E,6E-tridecadienoic acid C16 H26 O3 249.1857 14.5 93.94
123 Cucurbitacin A C32 H46 O9 579.2936 15.3 97.4
124 Graphinone C16 H24 O5 301.1414 15.3 99.32
125 Salicylic acid C7 H6 O3 121.0286 15.3 86.73
126 Lycopodine C16 H25 N O 248.2011 15.44 85.77
127 Oblongolide C14 H20 O2 243.1361 15.44 86.01
128 Botrydial C17 H26 O5 315.1574 16.06 96.63
129 Didrovaltratum C22 H32 O8 425.2154 16.57 92.54
130 Corrinoid C19 H22 N4 329.1732 16.8 85.29
131 Stigmatellin Y C29 H40 O6 485.2909 16.96 91.61
132 PE(20:4(5Z,8Z,11Z,14Z)/15:0) C40 H72 N O8 P 725.5193 17.66 86.02
133 Gymnemic acid I C43 H66 O14 827.3983 17.77 86.58
134 MG(0:0/22:4(7Z,10Z,13Z,16Z)/0:0) C25 H42 O4 406.33 19.13 93.72
135 trans-brassidic acid C22 H42 O2 338.3425 19.13 97.62
136 1b,3a,12a-Trihydroxy-5b-cholanoic acid C24 H40 O5 413.2669 20.82 98.56
137 3,4-dihydroxymandelate C8 H8 O5 167.034 20.83 87.44
138 Octanol C8 H18 O 113.1327 20.83 87.3
139 Di(2-ethylhexyl) adipate C22 H42 O4 393.2978 20.87 98.64
Phytochemicals present in herbal drug composition were separated and identified using LCMS- Agilent 6550 UHD Accurate Mass QTOF MS system. An instrumental specifications were as Mass Analyzer: Quadrupole Time Of Flight (QTOF), Ionisation Sources: ESI, Detector: Electron multiplier, Mass accuracy within 1 ppm; m/z (z=1) less than 500, MS/MS accuracy within 2 ppm, S/N ratio of 450:1, Resolution of 42,000 at m/z 2722, High Femtogram level sensitivity, 1pg at m/z 609, Dynamic range of up to five orders, Ability to scan multiple precursor ions ~50, Rapid polarity switching of 1.5 sec, Scan rates of 50 Hz and Positive modes. 20 ul of 1mg/ml Herbal drug composition prepared in MQ water was injected into the LCMS system with flow rate of 1ml/min of grading mobile phase of solvent Acetonitrile and water containing 0.1% Formic acid for 30 min. The total ion chromatogram (see figure 10) was obtained and mass spectrum of each peak was auto-analysed using plant metabolite reference library -Metlin, resulting to the identification of compounds present in herbal drug with probability above 85 percent as shown in table 1 below. These compounds individually or collectively may be responsible for the antisickling property of developed herbal drug composition.
Table 1: List of compounds detected in Herbal drug formulation by LCMS

Figure 10 shows the LCMS-Total Ion Chromatogram of the Herbal drug composition.
HPLC STUDY:
Liquid chromatography analysis of developed herbal drug composition was analysed using binary gradient HPLC instrument (Make-Shimadzu). 20 (microliter) ul of sample (2 mg/ml in water) was injected in HPLC systems and separated through C4 Column, 100Å, 5 µm, 4.6 mm X 250 mm using gradient mobile phase water (A) and acetonitrile (B), 5 min 5% B, 95%A; 25 min 95% B, 5% A; further till 30min 95% B, 5% A, then to 35 min 5% B, 95%A and kept constant up to 40 min. The chromatograms were derived at detection wavelength 254 nm and 366 nm and overlapped. The peak response at 254 nm was found optimum and relative peak area percentage was derived. The peak at 3.458 min RT showed 62.44 % abundance which can be a biomarker for the quality control of developed herbal drug. Said peak table is provided in table 2 below.
[0001] Table 2: Peak table – HPLC analysis
[0002]

Figure 11 shows the HPLC chromatogram of herbal drug composition.
HPTLC STUDY: HPTLC fingerprint of antisickling herbal drug was performed on Camag HPTLC instrument. 2mg per ml sample prepared in methanol, applied on TLC plate and developed using mobile phase methanol: ethyl acetate: glacial acetic acid (8:3:0.5 v/v/v). The TLP plate was visualized in UV chamber at 254 nm and image was captured as shown in figure 12. It can be used for quality control of developed herbal drug.
In another embodiment in-vivo acute and sub-acute toxicity study on rat model is tested for biosafety of the herbal drug composition.

TOXICOLOGICAL STUDY:
In-vivo preclinical biosafety of herbal drug composition disclosed herewith is performed in accordance with recommendations and approval of the Institutional Animal Ethical Committee of Agharkar Research Institute, Pune, India (IAEC APPROVAL NO.: ARI/IAEC/2019/14) at in-house Animal Toxicity Facility of Agharkar Research Institute, Pune, MH, India. SCDM refers to the antisickling herbal drug composition sample
Oral Acute Toxicity Study:
Male and female Wistar rats 6–8 week old (180–200?gm) were used in this study. Inbred Wistar strain of rats was housed at standard environmental conditions. They were fed on standard chow and provided purified water ad libitum.
Number of animals and dose levels

Sr. No. Group Code Group Number of
Animals[M+F] Days of experiment
1 (A) Control group I
(vehicle only) 03+03=06 14
2 (B) Treatment Group I (SCDM-I 2000mg/kg) at the time of feeding 03+03=06 14

Administration of doses: The test substance was administered in a single dose by gavage using oral feeding needle. Animals were fasted prior to dosing. After the substance has been administered, food was withheld for a further 3-4 hours in rats.
Observation:
Mortality- To be observed on 24, 48, & 72 hours
Clinical signs- Monitoring of convulsions, lethargy, sleep, coma, salivation, diarrhoea, skin colour, fur, eyes & mucus membrane
Necropsy- In case of animal dies
Duration- 14 Days
Sub acuteToxicity Study:
Housing and feeding conditions:
Inbred Wistar strain of rats was housed at standard environmental conditions. They were fed on standard chow and provided purified water ad libitum.
Number of animals and dose levels

Sr. No. Group Code Group Number of Animals[M+F] Days of experiment
1. (C) Control group II (vehicle only) 5+5=10 28
2. (D) Treatment Group II (SCDM-I 250 mg/kg) at
the time of feeding 5+5=10 28
3. (E) Treatment Group III (SCDM-I 500 mg/kg)
at the time of feeding 5+5=10 28
4. (F) Treatment Group IV (SCDM-I 1000 mg/kg)
at the time of feeding 5+5=10 28

Route of administration: Oral; Duration: 28 Days

Pre-experimental phase:

Acclimatization of animals
Period – 7 days (Recording of body weight and food intake twice in a week)
Urine qualitative test (Ames multiple sticks)
Fecal consistency (Filter paper technique)

Experimentation phase
Test compound exposure multiple dose (once daily for 28 days) and dosage schedule (as recommended by sponsor)
Mortality 6/12/24 hours
Body weight (Twice in a week)
Food consumption (Daily)
Cage side activity
Neurological examination
Urine qualitative test
Haematology –Hb, RBC, WBC, Platelet count, differential count
Clinical chemistry – albumin, total protein, serum creatine, SGOT, SGPT
Histopathology – Liver, and Kidney
Biochemical and histological analysis:
The blood was anaysed for hematological parameters using automated hematoanalyzer. The serum transaminase,creatine ,total protein ,cholesterol and bilirubin levels were analyzed by an automated biochemical analyzer using diagnostic kits (ERBA, Czech Republic). The histology was performed using standard protocol and the tissues were stained using hematoxylin and eosin (H&E).
Statistical analysis:
All statistical analyses were performed by using GraphPad Prism version 6 . Values are expressed as a mean?±?standard error of the mean (SEM). Data were analyzed using ANOVA and multiple comparisons were carried out using the Bonferroni post-hoc test. Statistical significance was considered for p values?32 I.U/L) 21.6 + 3.86 24.8 + 4.71
SGPT (>37 I.U/L) 20.16 + 1.69 17.91 + 1.23
13. Total bilirubin (0.2-0.5 mg/dl) 0.24 + 0.04 0.34 + 0.07
Serum creatinine (0.2-0.8 mg/dl) 0.28 +0,06 0.29 +0.04
14. Total cholesterol ( 90-150 mg/dl) 104.92 + 5.36 113.67 + 9.41

Histopathological Analysis
Oral Acute Toxicity Study
Table 5: Histopathological Observations of kidney and liver sections after oral acute toxicity

Histopathology Report of Acute Toxicity Study
Sr No. Group- Animal Code Histopathological Observations –
1. LIVER TISSUE Overall Pathological grade
1 Gr 1-
1 Normal histomorphological cellular features of hepatocytes with intact nucleus and cell borders. Absence of inflammatory or pathological changes in hepatic
parenchyma. NAD
2 Gr 1 -
2 Normal histomorphological cellular features of hepatocytes with intact nucleus and cell borders.
Absence of inflammatory or pathological changes in hepatic parenchyma. NAD
Gr 1-
3 Normal histomorphological cellular features of hepatocytes with intact nucleus and cell borders.
Absenceof inflammatory or pathological changes in hepatic parenchyma. NAD
4 Gr 1-
4 Normal histomorphological cellular features of hepatocytes with intact nucleus and cell borders.
Absence of inflammatory or pathological changes in hepatic parenchyma. NAD
5 Gr 1-
5 Normal histomorphological cellular features of hepatocytes with intact nucleus and cell borders.
Absence of inflammatory or pathological changes in hepatic parenchyma. NAD
6 Gr 1 –
6 Normal histomorphological cellular features of hepatocytes with intact nucleus and cell borders. Focal presence of degenerative changes of hepatocytes with vacuolar cytoplasmic changes.
Absence of inflammatory or pathological changes in hepatic parenchyma. Minimal (+1)
7 Gr 2 –
1 Normal histomorphological cellular features of hepatocytes with intact nucleus and cell borders.
Absence of inflammatory or pathological changes in hepatic parenchyma. NAD
8 Gr 2 –
2 Normal histomorphological cellular features of hepatocytes with intact nucleus and cell borders. Focal presence of degenerative changes of hepatocytes with vacuolar cytoplasmic changes.
Absence of inflammatory or pathological changes in hepatic parenchyma. Minimal (+1)
9 Gr 3 –
6 Normal histomorphological cellular features of hepatocytes with intact nucleus and cell borders.
Absence of inflammatory or pathological changes in hepatic parenchyma. NAD
10 Gr 4 –
6 Normal histomorphological cellular features of hepatocytes with intact nucleus and cell borders. Focal congested vascular tissue in hepatic parenchyma. Absence
of inflammatory or pathological changes in hepatic parenchyma. NAD
11 Gr 5 –
6 Normal histomorphological cellular features of hepatocytes with intact nucleus and cell borders. Focal congested vascular tissue in hepatic parenchyma. Absence
of inflammatory or pathological changes in hepatic parenchyma. NAD
12 Gr 6 –
6 Normal histomorphological cellular features of hepatocytes with intact nucleus and cell borders.
Focal congested vascular tissue in hepatic parenchyma.
Absence of inflammatory or pathological changes in hepatic parenchyma. NAD
Note : Overall Grade score as- NAD =No Abnormality Detected, Minimal changes (+1), Mild changes (+2),
Moderate changes (+3), Severe changes (+4).

Sr No. Group / Animal Code Histopathological Observations –
2. Kidney TISSUE Overall Pathological grade
1 Gr 1 – 1 Normal histomorphological features of renal parenchyma with intact nucleus and cell borders of renal tubules and glomeruli. Absence of inflammatory or pathological changes in renal parenchyma. NAD
2 Gr 1 – 2 Normal histomorphological features of renal parenchyma with intact nucleus and cell borders of renal tubules and glomeruli. Absence of inflammatory or pathological changes in renal parenchyma. NAD
3 Gr 1 – 3 Normal histomorphological features of renal parenchyma with intact nucleus and cell borders of renal tubules and glomeruli. Absence of inflammatory or pathological changes in renal parenchyma. NAD
4 Gr 1 – 4 Normal histomorphological features of renal parenchyma with intact nucleus and cell borders of renal tubules and glomeruli. Absence of inflammatory or pathological changes in renal parenchyma. NAD
5 Gr 1 – 5 Normal histomorphological features of renal parenchyma with intact nucleus and cell borders of renal tubules and glomeruli. Absence of inflammatory or pathological changes in renal parenchyma. NAD
6 Gr 1 – 6 Normal histomorphological features of renal parenchyma with intact nucleus and cell borders of renal tubules and glomeruli. Focal congested vascular tissue in medulla region. Absence of inflammatory or pathological changes in renal parenchyma. NAD
7 Gr 2 – 1 Normal histomorphological features of renal parenchyma with intact nucleus and cell borders of renal tubules and glomeruli. Absence of inflammatory or pathological changes in renal parenchyma. NAD
8 Gr 2 – 2 Normal histomorphological features of renal parenchyma with intact nucleus and cell borders of renal tubules and glomeruli. Absence of inflammatory or pathological changes in renal parenchyma. NAD
9 Gr 2 – 3 Normal histomorphological features of renal parenchyma with intact nucleus and cell borders of renal tubules and glomeruli. Absence of inflammatory or pathological changes in renal parenchyma. NAD
10 Gr 2 – 4 Normal histomorphological features of renal parenchyma with intact nucleus and cell borders of renal tubules and glomeruli. Absence of inflammatory or pathological changes in renal parenchyma. NAD
11 Gr 2 – 5 Focal congested blood vessels in medullary region.
Normal histomorphological features of renal parenchyma with intact nucleus and cell borders of renal tubules and glomeruli. Absence of inflammatory or pathological changes in renal parenchyma. NAD
12 Gr 2 – 6 Normal histomorphological features of renal parenchyma with intact nucleus and cell borders of renal tubules and glomeruli. Absence of inflammatory or pathological changes in renal parenchyma. NAD

The photomicrographs of kidney and liver sections (H & E staining) after acute oral toxicity is shown in figure 13
Sub Acute Toxicity Study:
Table 6: Histopathological Observations of kidney and liver sections after oral subacute toxicity

Sr No. Group- Animal
Code Histopathological Observations –
1. LIVER TISSUE Overall Pathological
grade
1 Gr 1
– 1 Normal histomorphological cellular features of hepatocytes with intact nucleus and cell borders. Absence of inflammatory or pathological changes in hepatic
parenchyma. NAD
2 Gr 1 –
1 Normal histomorphological cellular features of hepatocytes with intact nucleus and cell borders.
Absence of inflammatory or pathological changes in hepatic parenchyma. NAD
3 Gr 1 –
3 Normal histomorphological cellular features of hepatocytes with intact nucleus and cell borders.
Absence of inflammatory or pathological changes in hepatic parenchyma. NAD
4 Gr 1 –
4 Normal histomorphological cellular features of hepatocytes with intact nucleus and cell borders.
Absence of inflammatory or pathological changes in hepatic parenchyma. NAD
5 Gr 1 –
5 Normal histomorphological cellular features of hepatocytes with intact nucleus and cell borders.
Absence of inflammatory or pathological changes in hepatic parenchyma. NAD
6 Gr 1 –
1 Normal histomorphological cellular features of hepatocytes with intact nucleus and cell borders.
Absence of inflammatory or pathological changes in hepatic parenchyma. NAD
7 Gr 2 –
2 Normal histomorphological cellular features of hepatocytes with intact nucleus and cell borders.
Absence of inflammatory or pathological changes in hepatic parenchyma. NAD
8 Gr 2 –
3 Normal histomorphological cellular features of hepatocytes with intact nucleus and cell borders. Focal presence of degenerative changes of hepatocytes with vacuolar cytoplasmic changes.
Absence of inflammatory or pathological changes in hepatic parenchyma. Minimal (+1)
9 Gr 2 –
4 Normal histomorphological cellular features of hepatocytes with intact nucleus and cell borders.
Absence of inflammatory or pathological changes in hepatic parenchyma. NAD
10 Gr 2 –
4 Normal histomorphological cellular features of hepatocytes with intact nucleus and cell borders. Focal congested vascular tissue in hepatic parenchyma. Absence
of inflammatory or pathological changes in hepatic parenchyma. NAD
11 Gr 2 –
5 Normal histomorphological cellular features of hepatocytes with intact nucleus and cell borders.
Absence of inflammatory or pathological changes in hepatic parenchyma. NAD

12 Gr 2 –
6 Normal histomorphological cellular features of hepatocytes with intact nucleus and cell borders.
Focal congested vascular tissue in hepatic parenchyma.
Absence of inflammatory or pathological changes in hepatic parenchyma. NAD
13 Gr 3–
1 Normal histomorphological cellular features of hepatocytes with intact nucleus and cell borders. Absence of inflammatory or pathological changes in hepatic
parenchyma. NAD
14 Gr 3 –
1 Normal histomorphological cellular features of hepatocytes with intact nucleus and cell borders. Focal presence of degenerative changes of hepatocytes with vacuolar cytoplasmic changes.
Absence of inflammatory or pathological changes in hepatic parenchyma. Minimal (+1)
15 Gr 3 –
3 Normal histomorphological cellular features of hepatocytes with intact nucleus and cell borders.
Absence of inflammatory or pathological changes in hepatic parenchyma. NAD
16 Gr 3 –
4 Normal histomorphological cellular features of hepatocytes with intact nucleus and cell borders.
Absence of inflammatory or pathological changes in hepatic parenchyma. NAD
17 Gr 3 –
5 Normal histomorphological cellular features of hepatocytes with intact nucleus and cell borders.
Absence of inflammatory or pathological changes in hepatic parenchyma. NAD
18 Gr 3 –
6 Normal histomorphological cellular features of hepatocytes with intact nucleus and cell borders.
Absence of inflammatory or pathological changes in hepatic parenchyma. NAD
19 Gr 4 –
1 Normal histomorphological cellular features of hepatocytes with intact nucleus and cell borders.
Absence of inflammatory or pathological changes in hepatic parenchyma. NAD
20 Gr 4 –
2 Normal histomorphological cellular features of hepatocytes with intact nucleus and cell borders.
Focal congested vascular tissue in hepatic parenchyma.
Absence of inflammatory or pathological changes in hepatic parenchyma. NAD
21 Gr 4 –
3 Normal histomorphological cellular features of hepatocytes with intact nucleus and cell borders.
Absence of inflammatory or pathological changes in hepatic parenchyma. NAD
22 Gr 4 –
4 Normal histomorphological cellular features of hepatocytes with intact nucleus and cell borders. Focal congested vascular tissue in hepatic parenchyma. Absence
of inflammatory or pathological changes in hepatic parenchyma. NAD
23 Gr 4–
5 Normal histomorphological cellular features of hepatocytes with intact nucleus and cell borders. Focal presence of degenerative changes of hepatocytes with vacuolar cytoplasmic changes.
Absence of inflammatory or pathological changes in hepatic parenchyma. Minimal (+1)
24 Gr 4 –
6 Normal histomorphological cellular features of hepatocytes with intact nucleus and cell borders.
Absence of inflammatory or pathological changes in hepatic parenchyma. NAD

Sr No. Group / Animal Code Histopathological Observations –
2. Kidney TISSUE Overall Pathological grade
1 Gr 1 –
1 Normal histomorphological features of renal parenchyma with intact nucleus and cell borders of renal tubules and glomeruli. Absence of inflammatory or pathological changes in renal parenchyma. NAD
2 Gr 1 – 1 Normal histomorphological features of renal parenchyma with intact nucleus
and cell borders of renal tubules and glomeruli. Absence of inflammatory or pathological changes in renal parenchyma. NAD
3 Gr 1 – 3 Normal histomorphological features of renal parenchyma with intact nucleus and cell borders of renal tubules and glomeruli. Absence of inflammatory or
pathological changes in renal parenchyma. NAD
4 Gr 1 – 4 Normal histomorphological features of renal parenchyma with intact nucleus and cell borders of renal tubules and glomeruli. Absence of inflammatory orpathological changes in renal parenchyma. NAD
5 Gr 1 – 5 Normal histomorphological features of renal parenchyma with intact nucleus and cell borders of renal tubules and glomeruli. Absence of inflammatory or
pathological changes in renal parenchyma. NAD
6 Gr 1 – 1 Normal histomorphological features of renal parenchyma with intact nucleus and cell borders of renal tubules and glomeruli. Absence of inflammatory or
pathological changes in renal parenchyma. NAD
7 Gr 2 – 2 Normal histomorphological features of renal parenchyma with intact nucleus and cell borders of renal tubules and glomeruli. Absence of inflammatory or pathological changes in renal parenchyma. NAD
8 Gr 2 – 3 Normal histomorphological features of renal parenchyma with intact nucleus and cell borders of renal tubules and glomeruli. Focal congested vascular tissue in medulla region. Absence of inflammatory or pathological changes in
renal parenchyma. NAD
9 Gr 2 – 4 Normal histomorphological features of renal parenchyma with intact nucleus and cell borders of renal tubules and glomeruli. Absence of inflammatory or
pathological changes in renal parenchyma. NAD
10 Gr 2 – 4 Normal histomorphological features of renal parenchyma with intact nucleus and cell borders of renal tubules and glomeruli. Absence of inflammatory or
pathological changes in renal parenchyma. NAD
11 Gr 2 – 5 Normal histomorphological features of renal parenchyma with intact nucleus and cell borders of renal tubules and glomeruli. Absence of inflammatory or
pathological changes in renal parenchyma. NAD
12 Gr 2 – 6 Normal histomorphological features of renal parenchyma with intact nucleus and cell borders of renal tubules and glomeruli. Absence of inflammatory or
pathological changes in renal parenchyma. NAD
13 Gr 3– 1 Normal histomorphological features of renal parenchyma with intact nucleus
and cell borders of renal tubules and glomeruli. Absence of inflammatory or pathological changes in renal parenchyma. NAD
14 Gr 3 – 1 Normal histomorphological features of renal parenchyma with intact nucleus and cell borders of renal tubules and glomeruli. Absence of inflammatory or
pathological changes in renal parenchyma. NAD
15 Gr 3 – 3 Normal histomorphological features of renal parenchyma with intact nucleus and cell borders of renal tubules and glomeruli. Focal congested vascular tissue in medulla region. Absence of inflammatory or pathological changes in
renal parenchyma. NAD
16 Gr 3 – 4 Normal histomorphological features of renal parenchyma with intact nucleus and cell borders of renal tubules and glomeruli. Absence of inflammatory or
pathological changes in renal parenchyma. NAD
17 Gr 3 – 5 Normal histomorphological features of renal parenchyma with intact nucleus and cell borders of renal tubules and glomeruli. Absence of inflammatory or
pathological changes in renal parenchyma. NAD
18 Gr 3 – 6 Normal histomorphological features of renal parenchyma with intact nucleus and cell borders of renal tubules and glomeruli. Absence of inflammatory or
pathological changes in renal parenchyma. NAD
19 Gr 4 – 1 Normal histomorphological features of renal parenchyma with intact nucleus and cell borders of renal tubules and glomeruli. Absence of inflammatory or pathological changes in renal parenchyma. NAD
20 Gr 4 – 2 Normal histomorphological features of renal parenchyma with intact nucleus and cell borders of renal tubules and glomeruli. Absence of inflammatory or
pathological changes in renal parenchyma. NAD
21 Gr 4 – 3 Normal histomorphological features of renal parenchyma with intact nucleus and cell borders of renal tubules and glomeruli. Focal congested vascular tissue in medulla region. Absence of inflammatory or pathological changes in
renal parenchyma. NAD
22 Gr 4 – 4 Normal histomorphological features of renal parenchyma with intact nucleus and cell borders of renal tubules and glomeruli. Focal congested vascular tissue in medulla region. Absence of inflammatory or pathological changes in
renal parenchyma. NAD
23 Gr 4– 5 Normal histomorphological features of renal parenchyma with intact nucleus and cell borders of renal tubules and glomeruli. Absence of inflammatory or
pathological changes in renal parenchyma. NAD
24 Gr 4 – 6 Normal histomorphological features of renal parenchyma with intact nucleus and cell borders of renal tubules and glomeruli. Absence of inflammatory or
pathological changes in renal parenchyma. NAD
Note : Overall Grade score as- NAD =No Abnormality Detected, Minimal changes (+1), Mild changes (+2),Moderate changes (+3), Severe changes (+4).
The photomicrographs of kidney and liver sections (H & E staining) after subacute toxicity study is shown in figure 14.
Interpretation:
Oral Acute Toxicity Study: An oral Acute Toxicity Study was performed for 14 days with 7 doses of drug as per standard approved protocol. The male and female Wistar rats available in the facility of Agharkar Research Institute, Pune MH were treated with the antisickling herbal drug composition at a dose of 2000?mg/kg of body weight. The results showed no mortality or any untoward symptoms or abnormal behavioural changes during the 14-day observation period by following the dosing. No adverse changes were observed in haematological, biochemical and histological parameters.
Sub-acute toxicity study was performed for 28 days. No mortality or any untoward symptoms or abnormal behavioural changes were observed in group of rats treated with 250, 500 and 1000 mg/kg with respect to control group. Furthermore, the haematological, biochemical and histological changes were not observed as compare to test group with any tested dose levels of herbal antisickling agent. This ensures that the developed herbal drug is safe to use clinically even at higher doses. This is supported by the photomicrographs of kidney and liver sections (H & E staining) after acute oral toxicity as shown in figure 13 and subacute toxicity study as shown in figure 14.
The foregoing description of the specific embodiments will so fully reveal the general nature of the embodiments herein that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. Therefore, while the embodiments herein has been described in terms of preferred embodiments, those skilled in the art will recognize that the embodiments herein can be practiced with modification within the spirit and scope of the application.
,CLAIMS:We Claim:

1. A herbal drug composition comprising of plant extracts of Thinopyrum intermedium (Wheatgrass), Punica granatum (Pomegranates peel), Rubia cordifolia (Indian madder stem) and Aegle marmelos (Indian bael leaf); wherein the Thinopyrum intermedium (Wheatgrass) extract is present at concentration ranging from about 10% w/w to about 40% w/w; the Punica granatum (Pomegranates peel) extract is present at concentration ranging from about 10% w/w to about 40% w/w; the Rubia cordifolia extract (Indian madder stem) is present at concentration ranging from about 10% w/w to about 40% w/w and Aegle marmelos extract (Indian bael leaf) is present at concentration ranging from about 10% w/w to about 40% w/w.

2. The herbal composition as claimed in claim 1, wherein the extract is an aqueous extract or any other solvent extract selected from the group consisting of methanol, ethanol, hydro-alcohol, ethyl acetate, acidified aqueous solution, saline water or buffer solutions and wherein the Thinopyrum intermedium (Wheatgrass) extract is present at concentration 12.50%, 25% or 37.5% (w/w); the Punica granatum (Pomegranate’s peel) extract is present at concentration 12.50%, 25% or 37.5% (w/w); the Rubia cordifolia (Indian madder stem) extract is present at concentration 12.50%, 25% or 37.5% (w/w) and Aegle marmelos (Indian bael leaf) extract is present at concentration 12.50%, 25% or 37.5% (w/w).

3. The herbal composition as claimed in claim 1, wherein the Thinopyrum intermedium (Wheatgrass) extract is present at concentration 25% (w/w); the Punica granatum (Pomegranate’s peel) extract is present at concentration 25% (w/w); the Rubia cordifolia (Indian madder stem) extract is present at concentration 25% (w/w) and Aegle marmelos (Indian bael leaf) extract is present at concentration 25% (w/w).

4. The herbal composition as claimed in claim 1, wherein the composition is in the form of tablets, capsules, syrup or any suitable form for oral administration and wherein the Human Equivalent Dose (HED) dose of the herbal drug composition ranges from about =120 to 1000 mg/dose and preferably from about =18 mg/kg to 150 mg/kg..

5. The herbal composition as claimed in claim 1, wherein the composition has good in-vitro polymerization inhibition of the Sickle hemoglobin protein (Hb S) and anti-sickling activity on sickled RBCs.

6. The herbal drug composition as claimed in claim 1, wherein the drug composition has good percentage of reversal of sickling of the sickled RBCs and antioxidant potential for protecting oxidative damage in the sickle cell disease (SCD).

7. The herbal drug composition as claimed in claim 1, wherein the each component of herbal drug composition has antisickling property and can act as antisickling agent.

8. A method of preparing a herbal drug composition, said method comprising the steps of:
i) collecting materials selected Thinopyrum intermedium (Wheatgrass), Punica granatum (Pomegranate’s peel), Rubia cordifolia (Indian madder stem), and Aegle marmelos (Indian bael leaf);
ii) preparing an extract of each plant material of step i) individually, wherein the extract is an aqueous extract.
iii) filtering the aqueous extract of each plant material obtained in step ii) to obtain a filtrate of each plant material;
iv) drying the filtrate of each aqueousextract of step iii) to obtain a solid plant extract of each plant material; and
v) combining the dried aqueous extracts of each plant material of step iv) in a suitable proportion to obtain a herbal drug composition.
9. A method of preparing a herbal drug composition, said method comprising the steps of:
i) collecting materials selected Thinopyrum intermedium (Wheatgrass), Punica granatum (Pomegranate’s peel), Rubia cordifolia (Indian madder stem), and Aegle marmelos (Indian bael leaf);
ii) preparing an extract of each plant material of step i) individually, wherein the extract is an extract of any other solvent selected from the group consisting of methanol, ethanol, hydro-alcohol, ethyl acetate, acidified aqueous solution, saline water or buffer solutions.
iii) filtering the extract of each plant material obtained in step ii) to obtain a filtrate of each plant material;
iv) drying the filtrate of each extract of step iii) to obtain a solid plant extract of each plant material; and
v) combining the dried extracts of each plant material of step iv) in a suitable proportion to obtain a herbal drug composition

10. The method of preparing a herbal drug composition as claimed in claims 8 or 9, wherein the extract of each plant material is prepared using decoction technique and wherein the filtrate of the plant extract is dried using spray drying, high vacuum evaporation or lyophilisation.

11. The method of preparing a herbal drug composition as claimed in claims 8, or 9 wherein the plant extract of Thinopyrum intermedium (Wheatgrass) is present at concentration ranging from about 10% w/w to about 40% w/w; the plant extract of Punica granatum (Pomegranates peel) is present at concentration ranging from about 10% w/w to about 40% w/w; the plant extract of Rubia cordifolia (Indian madder stem) is present at concentration ranging from about 10% w/w to about 40% w/w and the aqueous plant extract of Aegle marmelos (Indian bael leaf) is present at concentration ranging from about 10% w/w to about 40% w/w.

12. The method of preparing a herbal drug composition as claimed in claims 8, or 9 wherein the plant extract of Thinopyrum intermedium (Wheatgrass) is present at concentration 12.50%, 25% or 37.5% (w/w); the plant extract of Punica granatum (Pomegranates peel) is present at concentration 12.50%, 25% or 37.5% (w/w); the plant extract of Rubia cordifolia (Indian madder stem) is present at concentration 12.50%, 25% or 37.5% (w/w) and the plant extract of Aegle marmelos (Indian bael leaf) is present at concentration 12.50%, 25% or 37.5% (w/w).

13. The method of preparing a herbal drug composition as claimed in claims 8, or 9 wherein the plant extract of Thinopyrum intermedium (Wheatgrass) is present at concentration 25% (w/w); the plant extract of Punica granatum (Pomegranates peel) is present at concentration 25% (w/w); the plant extract of Rubia cordifolia (Indian madder stem) is present at concentration 25% (w/w) and the plant extract of Aegle marmelos (Indian bael leaf) is present at concentration 25% (w/w).

Dated this 05th Day of June 2020

Priyank Gupta
Agent for the Applicant
IN/PA-1454