Integrative Biomedical Research

Introduction

People in Malaysia prefer to use traditional medicine before modern medicine is introduced. A complete report on the Malay traditional medicinal plants was compiled in a book, entitled “A Dictionary of the Economic Products of the Malay Peninsula”. This book has inspired both phytochemists and ethno-botanists to conduct research on medicinal plants, which   helps   add   knowledge   to   Malaysian   medicinal   plants   (R. Renuka et al., 2001, Al-Sokanee et al., 2009). F. deltoidea and G.sublanceolata leaves are used as traditional medicine. These plants are used to treat various     sickness and diseases. Malaysia is famous for F.deltoidea called “Mas Cotek”.  This plant belongs to Moraceae family.  F.deltoidea leaves have two kinds: female and male. Female leaves have higher active ingredients, so this kind of leaves are used for medical purposes compared to male ones.   Hail of Rubiaceae family includes G. Sublanceolata   leaves called Pitang.  These leaves turn brown when they are dry.  It is very difficult to find these leaves because they are very rare; however, we can find a huge number of these plants in Thailand. These plants have a diversity  of  Pigments,  namely  xanthophyll,    chlorophyll,  flavonol, flavones,  carotene and  anthocyanin  (Mucalo  et al., 2004). The biochemical ingredient of the plants has a significant role as the natural antioxidants and the activity of the extracted plant polymers is higher when they consumed as crude and the combination of different extracts (R. Renuka et al., 2001). However, the major disadvantage is that the quantity of herbal extract necessary for the treatment is higher because the degradation of different plant compounds such as alkaloids, flavonoids, phenols, steroids, and anthraquinon in the gastrointestinal tract.  It is due to the acidic pH in the stomach, which increase their breakdown, loss of the effect and increment with the duration of treatment with the decreased absorption of these compounds in the intestine (Al-Sokanee  et al., 2009 ). Currently, several researchers and studies are concentrated on encapsulation/capping of the plant extracts to increase the sustained release of active compounds in the intestine for the maximal absorption (Mucalo et al., 2004, Hench et al., 1991, Ivone 2001, Kavitha et al., 2017, Walum et al., 1998, Yehya et al., 2019, Gopinath et al. 2016). Recently, nanotechnological methods have activated the advanced delivery systems, which include the controlled drug delivery to the site of action by developing hydroxyapatite and other nanoparticles (Anniebell and  Gopinath, 2018, Suk et al., 2018. Hydroxyapatite [Ca10(PO4)6(OH)2] (HAp), is the main constituents of bone and teeth, a  biomaterial in view of its excellent bioactivity, biocompatibility, non-toxicity and non-inflammatory. HAp nanoparticles can be prepared by the direct precipitation method possess the advanced important characteristics such as, large pore volumes, high surface area and reactive surfaces for post-functionalization and pure powder which make them ideal as potential carriers for the plant extract as drugs (Cao  et al., 2014).  Moreover, it has also been confirmed that HAp nanoparticles with the size lesser than 100 nm could be up taken by the cells efficiently. The biocompatibility and bioactivity of HAp nanoparticles represent a choice for the controlled drug delivery (Matouskova  et al., 2016). Using nanotechnology with phyto-extracts has explored a beneficial strategy for herbal drugs including the enhancement of bioavailability, bioavailability, solubility, sustained delivery pharmacological activity,  physical and chemical degradation and protection from toxicity (Arunachalam et al., 2017, Theivasanthi et al., 2018).  This is the first study were conducted on the ingestion of these encapsulated combine of plant extract  with  nanoHAp  at  high  doses.  Therefore,  there  is  a  need  to  explore the systemic for  evaluating  their  efficacy  and  safety  properties.  Hence,  this  study aims  to evaluate the safety of this extract with acute and sub-chronic toxicity tests in BALB/c mice model.

Materials and methods

Samples Collection and Extraction                                                                                                                                                

Ficus deltoidea (female leaves) and Gynochthodes sublanceolata were collected from the covered green house garden of School of Bioprocess Engineering and University Malaysia Perlis. Then, they were cleaned with a running tap water to remove debris and contamination. Each set of  the collection was dried at room temperature for two weeks. The dried leaves were ground, then 100 grams (50 grams from each plant leaves)  were mixed grams were mixed with 2000 ml of a methanol: distilled water (60:40 % v/v) was put in a beaker and covered with aluminum foil at an ambient temperature about 24 h and shaken during the extraction. The extract was filtered through Whatman No.1 filter paper. Then, the solvent was removed from samples using a rotary evaporator then freeze dryer was used to transform the sample into the powder form. Finally, the extract was placed in air-tight amber bottles and stored in a freezer to prevent the oxidation of damage until further use (Kavitha et al., 2017).

Hydroxyapatite (HAp) Preparation by direct precipitation

In this experiment, HAp was obtained by a direct precipitation. According to Song et al. the Stoichiometric reaction of calcium nitrate tetrahydrate (Ca(NO₃)_2. 4H₂O and ammonium phosphate (NH₄H₂PO₄) will yield the hydroxyapatite as below;

5Ca (NO₃) + 3(NH₄)₂ HPO₄+ H₂O ? Ca₅ (PO₄)₃OH+ 6NH₄NO₃+ 4HNO₃

Calcium nitrate tetrahydrate (Ca(NO₃)₂4H₂O and ammonium phosphate (NH₄H₂PO₄) were  dissolved in 100 ml distilled water at room temperature. In this experiment, calcium nitrate tetrahydrate act as a calcium source, while ammonium dihydrogen phosphate acts as a phosphorus source. The concentration 100 mM of (Ca(NO₃)₂4H₂O and 60 mM of (NH₄H₂PO₄) was applied to give the molar ratio of calcium to phosphate is 1.67. The solution then was poured and mix into a set up magnetic stirrer beaker on the hot plate at 60 °C while the stirring rate at 550 rpm the mixing was conducted for 1 hour. After that, the suspension will be filtered by using Whatman filter No.1 paper, the drying process was carried out in an oven at a temperature of 60 °C for 24 hours. Finally, the dried sample was crushed by using pestle and mortar for 5 minutes.

Preparation of Encapsulated Phyto-extract Nano-hybrid with Hap

Nanoparticles containing the phyto-components combination from the test plants were prepared by solvent evaporation method described before with the additional modifications (Bernard, S. A., &Olayinka, 2010. Accurately weighed amount of HAp and plant extract with mass ratio were chosen, which includes 1:5. They were separately dissolved in distilled water containing ethanol for plant extract and ethanol for HAp, then mixed and stirred for 2 hr to evaporate the organic solvent and left for 24 hr at room temperature. The nanoparticles formed were isolated by centrifugation for 15 minutes at 10000 xg. Finally, the nanoparticles were washed with deionized water to remove the residual solvent.

Test animal

The  animal  study  was  approved  and  conducted  in  strict  guidance  according to  Eman Research Animal Ethics Committee Malaysia (Reference #:     112074A2212130719. Healthy BALB/c mice in a weight of 20–30 g were kept in an animal house in EMAN Biodiscoveries, Malaysia.  Plastic cages (34 × 47 × 18 cm3) at animal house are used to keep   animals. There were five mice in each cage which is in an air conditioned environment at room  temperature of (25 ± 2)°C with relative humidity (60% ± 10%) under 12 h night and light cycle. The animals were fed with commercially available standard pellet chow and unlimited supply of filtered drinking water.

Acute toxicity study

The  oral  acute  toxicity  study  of   encapsulated   extract  was  evaluated     according  to Organization for Economic Cooperation and Development (OECD) guideline 423 on BALB/c mice (20–30 g) (Walum, 1998), where the test doses of 300, 2000 and 4000 mg/kg were used.   Before the experiment was done all the animals free excess to water, andwere kept at overnight fasting. The mice were divided into four groups of five animals for each group each (n=5). The 1st  group   belongs to the control, whereas the 2nd, 3rd and 4th  groups are experimental groups that   received orally encapsulated extract (in normal saline) at dose of 300 mg/kg, 2000 mg/kg and 4000 mg/kg respectively. Before dose administration, the body of  each animal was    weighed, and there was a calculation of the dose based on the body weight An animal was not observed any toxic effect for the first 4 hour after a period of treatment More animals    were observed and investigated  within 3  days  for  if any toxic effect. It  was  found  that  there were behavioral changes  and other  parameters  such as body weight,  urinations,  food intake,    water intake, respiration,  convulsion,  tremor,  temperature,  constipations.  Also,  their eye and skin colors changed etc (Yehya et al., 2019).                                        

Sub-chronic toxicity study

According  to  OECD  guideline  407  (Gopinath  et al., 2016),  oral  sub-chronic  toxicity  study   was  conducted. Fifteen healthy BALB/c mice (20–30 g) were divided into three groups five animal   each and kept   under   standard  conditions.   The  control  group   belongs   to  Group   I; whereas  the experimental  groups  are the other  two groups  which received the encapsulated  extract at a dose of  600 and 1000 mg/kg,  respectively within 28 consecutive days (Anniebell, and Gopinath, 2018). The control group was received normal saline. The second and third groups were given with a single dose of 600 mg/kg and 1000  mg/kg  of  body weight  of  encapsulated  extract, respectively. Gavage dosing was performed using a curved, ball-tipped intubation  needle affixed to a 5 ml syringe. Fresh preparation of solutions was made prior to dosing and they were kept chilled and tightly capped.

Hematological and biochemical examination

At  the end of study, animals  should be anaesthetized  with a ketamine and xylazine with 0.05-0.1mL/10g body weight . 1mL of ketamine (100mg/mL) and  0.5mL xylazine (20mg/mL) single dose.  after anovernight fasting (8 h).  Test tube contains blood sample with and  ethylene diaminetetra   acetic   acid  as  an  anticoagulant, and without it  respectively   for   biochemical   and  hematological parameters.  Biochemical  analysis  was  done  through  the  evaluation  of  blood without the ethylene diamine tetra acetic acid, allowed to clot after centrifugation at 2 500 r/min for 15 min to obtain serum and stored at -20°C until it was assayed for biochemical estimation.  After blood  was  collected,  all important  organ,  namely liver,  kidney, lung,  heart,  pancreas and small intestine were harvested. Each organ was weighed on electronic balance to  observe any changes in organs weights of treated animals compared to control group )( Suk  et al., 2018). The relative organ weight (ROW) of each organ was calculated as follows (Anniebell, and Gopinath, 2018):

ROW= (Absolute organ weight (g) / mice body weight on sacrifice day) ×100                                                                       

Effect of encapsulate plant extract on hematological parameters

Red blood cell count,  hematocrit,  mean  cell volume,  hemoglobin,  white blood cell count,  mean  corpuscular  hemoglobin  concentration,  mean  corpuscular volume,  monocyte, neutrophil, lymphocyte and platelet count of the control and treated groups   were determined and compared with control group  using an automatic  hematology analyzer (Sysmex K21, Tokyo, Japan).

Effect of encapsulate plant extract on serum biochemical parameters

There was an biochemical analysis on serum after collected blood was centrifugated, and the following parameters such as aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase, high density lipoprotein, total bilirubin (TBIL), total protein, albumin,   urea and creatinine level were determined for both control and extract treated groups. All  analyses were done through the analyzer of clinical chemistry.           

Histopathological studies

After the experimental stage, the animals  were sacrificed and necropsied.   Different visceral organs  including  kidney,  liver  and  lung  were  collected  and  fixed  in  10%  formaldehyde solution.  The  tissues  were  further  processed  with  the  Automatic  Tissue    Processor  and sectioned  at  5  µm  thickness  using  the  Rotary  Microtome  (Heitz  150 Rotary Microtome, Cambridge model)  and  embedded  in paraffin wax  to prepare blocks.  Sections were stained according   to   Haematoxylin   and   Eosin   (H    and    E)    technique   for microscopically examination (Suk et al., 2017). Subsequently,   the   sections   were   examined   using     Swift   Binocular Microscope  with  in-built  lighting  system  and  photographed  using  a  microscope-digital-camera with an Olympus photomicroscope.

Statistical analysis

Statistical analysis was done as mean of variance ± SEM (n = 5), followed by ANOVA test using Graph Pad Prism and for multiple comparison test among the groups, Bonferroni test was performed. A probability level 0of p < 0.001 was accepted statistically (Cao et al. 2014).

Results

Acute toxicity study

The acute toxic effect of encapsulated combination extracts was determined as per  the OECD guideline 423, where the limit test dose of 4000 mg/kg was used. No treatment related toxic symptom  or  mortality  were  observed  after  oral  administration  of  the tested encapsulated combination  extracts at a  dose of  300,  2000 and 4000  mg/kg.  The behavior  of  the treated groups with encapsulated combination extracts and the control group was first observed  for a short period (4 h), followed by a long period (72 h). The results showed no changed behavior, breathing, skin effects, water consumption, impairment in food intake and temperature.  Therefore, the extract is safe at a dose level of 4000 mg/kg, and the LD50 was >4000 mg/kg.   161 The parameters were observed for acute toxicity study after the test plant extract was 162 administered compared to the control group (Table 1).

Sub-chronic toxicity study

The sub-chronic toxic study on the tested plant extract was conducted as per OECD guidelinen407. All the tested group animals treated with encapsulated extract at a dose of 600 and 1000 mg/kg daily survived throughout the 28 days. There were no clinical toxicity signs in the experimental group compared to the control.                                                                                                                                                 

Effect of extract on relative organ body weight 

Average organs, body weight  and relative organs  weight  between the control group an encapsulated combine extracts treated group were no significantly different  at a dose of 600 and 1000 mg/kg. There  was  no  effect  of  tested  encapsulated  extract  on principal relative organto body weight (Tables 2 , 3,4). The weight was no significantly different. The results showed that the vital organs such as liver, kidney, heart, pancreas  and small  intestine  were  not  adversely affected throughout  the treatment   by the encapsulated combine extracts. There was no statistically significant    difference between the average and relative organ weight of control group and treated groups (p > 0.05).

Effect of encapsulated combine plants extract on hematological parameters

Table 5 show the results of the hematological tests. All the tested hematological parameters  such as total blood count, hemoglobin, red blood cell, total white blood cell, neutrophil, monocyte, lymphocyte, packed cell volume, and platelet count  of  the treated groups  were   within normal limits compared to the control group. There was no toxicologically significant difference (P > 0.05) between treated animals with the encapsulated combination extract and  control. The hematological parameters between the control and treated groups were not  significantly different.

Effect of encapsulated combine plants extract on biochemical parameters

Table  6  shows  the  results  of  the  various  biochemical  tests  on  the experimentally treated animals   with  the  plant   extract  and  the  control   group.   There  was   no   impact  of  oral administration  of  the  encapsulated  treated  at  a  dose  of  600  and  1000  mg/kg  on serum biochemical  parameters  such  as  albumin,  total  protein,  globulin,  T-BIL,    urea,  sodium, creatinine and uric acid levels and SGOT (AST) and SGPT (ALT)  when compared to control group. There was no significant                           

Table 5  values are expressed as mean ± S.E.M. P > 0.05  when  compared  to  normal  control  group.  226 PCV=packed cell volume, MCV=Mean cell volume or mean corpuscular volume, MCH= Mean cell 227 haemoglobin and MCHC= mean cell haemoglobin concentration and WBC=white blood cells. Statistical comparisons were made within a column and values with the same superscript letter are not significantly different.

Histology of selected organs

Histopathological examination of formalin fixed paraffin embedded tissues of liver    (Fig. 1), kidney  (Fig.  2),  and  lung  (Fig.  3)  of  all  treatment  groups  and  negative   control  group. Histopathology studies showed no adverse effect and potential of    encapsulated combination extracts.  The result  suggests  that  there  is  no  histopathological  abnormality  in the tested 9 groups. The effect of encapsulated combination extracts was investigated in   selected tissues. The H  &  E  sections  were studied under  photomicrographs  was  performed  at  100  x  magnification using Olympus light microscope.

Discussion

This  study  aimed  to  evaluate     the  encapsulation combination of F.deltoidea and G.sublanceolata leaf extracts with a Nano  hydroxyapatite for exploring acute and sub-chronic toxicity and identifying the range of dose that could  be used for further studies. The oral acute toxicity of the tested plant extract was  explored on BALB/c mice at a single dose of  300, 2000 and 4000 mg/kg body weight, and it was monitored for the first 4 h, followed for a period of 72 h for any toxic effect after a period of treatment. The results  revealed no  major  changes  in behavior,  mortality  and appeared normal  for general anatomical appearance in all groups). The encapsulated extract don't showed any toxic at a dose level of 4000 mg/kg, and the LD50 is >4 000 mg/kg when used with sub-chronic study. Critical toxicological effects such as teratogenesis  and reproductive disruption  have not been explored. Therefore need more investigations on toxic effects. (Arsad et al., 2018). It is suggested that the encapsulation extract is practically non-toxic in a single dose of level 4000 mg/kg body weight. However, study on   sub –chronic toxicity showed that multiple doses is used to treat chronic disorder such as    cancer, diabetes or hyperlipidemia safely. It does not affect the weight of relative organ, hematological and biochemical parameters. Study on a sub-acute toxicity was done with a dose of 600  and 1000 m g/kg of  extract as per  OECD guideline (Donko et al., 2014).  There was a toxic effect of chemicals and   289  drugs on decreasing or increasing the weight of body. However, scientific evidence showed  increase or decrease in the weight of body when fat  is  accumulated and  physiological   adaptation responds   the extract of plants rather than the toxic effects of chemicals or  drugs      that lead to decreasing appetite, hence, lower caloric intake by the animal (Kausar et al., 2010). The relative  weight of the vital organs, namely liver, kidney, heart,  pancreas  and small  intestine  was   normal, which indicated that there was no significant difference in toxic effect between the control and the experimental group (p > 0.05). The results show that this  no significant difference in the liver,  kidney,  heart and small  intestine   weight and in general anatomical appearance (Figure 1). After 28 days of treatment with testedextract, the hematological parameters were not significantly different, at p > 0.05, compared  to the control group.

The bone marrow impacts  the production of blood cell,  and     some phytochemicals isolated from plants affect the level of blood cell. Thus, the extract of tested plant does  not affect the function of bone marrow.  It  was  indicated that  all  doses  of  encapsulate extract do not induce anemia, making it safe. Similarly, the estimation of serum biochemical   parameters in treated  animals  was  not  significantly  different  (p  >  0.05),  compared  to  the  control  group. When the plasma membranes of liver  cells are damaged,  various enzymes in the   cytosol are released  into  the  stream   of  blood.  The extent  and  type  of Hepatocellular   damage  is quantitatively  measured  based  on  the levels  of  these  enzymes  in  the serum.  The  lack of alteration in liver  parameters  (the transaminases  enzyme SGOT  (AST) and     SGPT (ALT) showed that the administration of extrat for 28 days is not any abnormalities to the liver. The results also showed that the indicators of kidney tests (creatinine, uric acid, urea,    albumin, globulin,     bilirubin, and total protein) are not affected. Thus, livers or kidneys are not damaged by the sub-chronic administration of extract.  Researcher reported that no  overt signs of acute toxicity or death were observed in mice and rats treated with a methanol extract of F. deltoidea up to the dose of 6400 mg/kg (Ilyanie  et al., 2011).

However, the differential leukocyte counts for monocyte and eosinophils around within the reference value range (Harkness et al., 1993), which strongly suggests that there is no effects to treatment with encapsulated combination extracts. All biochemical parameters analyzed remained within the  reference levels for the species (Elham Farsi et al., 2013). Beside that these results take indicate on adverse effects of   gynocthodes sublanceolata and hydroxyapatite. The histopathological examination of selected  organs (liver, kidneys and lung) harvested from treated and control animals  confirm these results. This analysis revealed normal architecture for these vital organs. In the  liver  parenchyma of animals treated with extrat at doses up to 1000 mg/kg, normal-sized cells with     a centrally located euchromatic nucleus and a very prominent nucleolus were observed. The  hepatic vascular distribution was homogeneous when compared with that of the control group (Figure 2) with a normal hepatic portal triad. The results showed no significant difference  between the kidney sections of the control and the experimental groups, where urinary pole, vascular pole, glomerulus, convoluted tubules are normal and clearly visible (Figure 3). The lungs, which showed mild irritation of  in the air spaces for  both the treated  group  with 1000, 600 mg/kg and control.   These morphological changes in the lungs were due to by the daily oral gavage ordue to the anesthetized procedure  and not by encapsulated extract itself because these changes were also showed in the control group.  All  vital organs have a normal histological architecture and do not have any precipitation of nano  hydroxyapatite (Rhiouaniet al., 2008,  Muhammad et al., 2015). The histological studies revealed no obvious detrimental effects or  morphological disturbances of the daily oral administration of extract for 28 days, even at the highest tested dose of 1000 mg/kg.

Conclusion

Overall,  this  study  provides  preliminary  scientific  evidence  about  the  safety    profile  of Encapsulation of  combine plants  extract  of  Ficus  deltoidea and Gynocthodes sublanceolata for  long-term use.  The result  confirms  that  the use  and  development  of  encapsulation of combine plants  extract as  a therapeutic  agent  seems  to  be  quite  safe.    The use of encapsulation of combine plants extract and its mechanism of safety are meeting requirements   on the level of approach chosen in this study which might be applied for future human pre-  clinical studies.Critical toxicological effects such as teratogenesis  and reproductive disruption  have not been explored. Therefore need more investigations on toxic effects.

Author Contributions

R.K.A. and M.S. run the in vitro assay, M.N. assisted with experimental protocol and drafted the paper, F.S.A.S. designed the study, drafted and revised the article.

Acknowledgment

Authors would like to acknowledge School of Bioprocess Engineering, University Malaysia Perlis for the identification of the plant.

References